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Synthetic peptide with antiproliferative activity: a short C-terminal fragment of the human interferon α-2 molecule
Immunology Letters, 31 (1991) 1 5 - 20 Elsevier IMLET 01706
Synthetic peptide with antiproliferative activity: a short C-terminal fragment of the human interferon o -2 molecule A. V. D a n i l k o v i c h l, K. V. F r e z e 1, A. F. Shevalier 2, V. V. S a m u k o v 3, A. F. K i r k i n I a n d M. V. Gusev l 1Department of Cellular Physiology and Immunology, Biological Faculty, Moscow State University, Moscow; 2M. M. Shemyakin Institute of Bioorganic Chemistry, Moscow, and 3All Union Research Institute of Molecular Biology, Koltsovo, Novosibirsk Region, U.S.S.R. (Received 25 January 1991; revision received 23 July 1991; accepted 25 July 1991)
1. Summary The biological activity of six synthetic peptides of the 124 - 144 region of the human interferon a-2 (IFN or-2) molecule was studied. Peptides were examined for their ability to inhibit mitogen induced proliferation of human blood cells in vitro. Only the peptide corresponding to the amino acid sequence 1 2 4 - 1 3 8 (2438) possessed IFN-like antiproliferative activity. Other tested synthetic peptides did not affect cell proliferation in this experimental system. As with the native IFN or-2 molecule, the inhibitory effect of the peptide 2438 was dose-dependent. On simultaneous addition of peptide 2438, antiproliferative activity of IFN or-2 was enhanced. Direct cytotoxic effects of synthetic peptide 2438 were not revealed. These results suggest that a synthetic peptide corresponding to the 1 2 4 - 138-amino acid sequence of the human IFN o~-2 molecule serves as a cytostatic agent.
2. Introduction H u m a n o~-IFNs are a family of proteins which includes numerous subtypes showing over 75% homology of amino acid sequences [1]. IFN-a exerts various biological activities in different cell types, Key words: IFN a-2; Synthetic peptide; Human; T lymphocyte; Proliferation Correspondence to: Alia V. Danilkovich, Department of Cellular Physiology and Immunology, Biological Faculty, Moscow State University, Lenin's Hill, Moscow 119899, U.S.S.R.
both in vivo and in vitro [2]. Streuli et al. [3] have postulated that the IFN-a molecule contains at least two epitopes which interact with the receptor, one of them being located in the N-terminus and the other in the C-terminus of the IFN-o~ molecule. It was shown that the N-terminal part of the IFN-a molecule manifests antiviral and antiproliferative activities, and binds to the cellular receptor as does the whole IFN molecule [4]. At present there are no experimental data unambiguously confirming the existence of C-terminal active sites similar to those located in the Nterminus of the IFN c~-2 molecule. Other data support the involvement of the C-terminus in biological functions of IFN: (i) the Cys138 amino acid residue of the human IFN a-2 molecule is known to form the S - S bond important for biological activity of IFN [5]; (ii) a model of human IFN-a tertiary structure predicts that the fragment 1 2 0 - 147 is located near the N-terminal active site of IFN [6]; (iii) the amino acid sequence 1 2 0 - 1 4 7 of the IFN-ot molecule is highly (more than 86%) homologous among all the members of the family [7]. In the present study, we used previously synthesized peptides of the 1 2 4 - 144 segment of the IFN a-2 molecule [8] and examined their effects on the cell blast transformation response induced by polyclonal mitogens such as phytohemagglutinin (PHA) and interleukin-2 (IL-2) in vitro. The results demonstrate that like the intact IFN molecule, peptide 2438 corresponding to the 1 2 4 - 138-amino acid sequence of human IFN or-2 molecule can inhibit proliferation of mitogen-stim-
0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
15
ulated blood leukocytes in vitro. 3. Materials and Methods
3.1. Synthetic peptides A list of synthetic peptides used in these experiments and their location within the COOH-terminal part of the human IFN a-2 molecule is shown in Table 1. Their synthesis was performed as described [8].
3.2. Separation of lymphocytes Mononuclear cells were separated from the blood of healthy donors according to the method of Boyum [9]. The adherent cells were removed by incubation on plastic Petri dishes for 1 h at 37 °C in an atmosphere of 5°7o CO 2. The suspension of lymphocytes was divided into adherent and non-adherent subsets on a nylon wool column [10]. The nonadherent fraction was eluted with RPMI-1640 medium supplemented with 5°7o heat-inactivated fetal calf serum. This fraction contains T lymphocytes and null cells with small amounts of monocytes and B lymphocytes. 3.3. Proliferation assay Mononuclear cells, or separated T lymphocytes, were cultured in RPMI-1640 medium (Sigma) supplemented with 5°70 heat-inactivated fetal calf serum (Sigma), 100mM L-glutamine, 50/~g/ml gentamycin and 10/~M 2-mercaptoethanol in a humid atmosphere containing 5°70 CO 2 at 37 °C. 200/~l of lymphocyte suspension (mononuclear
cells or separated T-lymphocytes), at a concentration of 106 cells/ml in a 96-well flat-bottomed plate, were cultured in media containing P H A (2/~g/ml) or IL-2 (20 U/ml) in the presence of IFN o~-2 or synthetic peptides or IFN plus peptide for 75h. For the last 15h of culture, [3H]thymidine (0.5 tzCi/well; Amersham Corporation) was added. Cells were harvested and their radioactivity measured in a liquid scintillation counter (Rack-Beta LKB-Wallac, Sweden). P H A was obtained from Serva Laboratories. H u m a n recombinant IFN a-2 and human recombinant IL-2 were kindly provided by Dr. G. Chipens from the Institute of Organic Synthesis, Riga, Latvia, U.S.S.R. 4. Results and Discussion
Numerous experimental results emphasize the importance of the COOH-terminal part of the IFNot molecule in its biological action [11 - 14]. Earlier, six peptides of the 1 2 4 - 144 region of the human IFN a-2 molecule were synthesized (Table 1), and their antiviral potential was examined. It was found that, in contrast to the native IFN molecule, these peptides did not reveal antiviral activity [8]. This result agrees with the data on antiviral activity of different peptides from the COOHterminus of IFN-et molecules [15 - 17]. Interestingly, rabbit antisera developed against peptides 1 2 4 - 138 and 1 2 9 - 144 showed an ability to bind recombinant IFN-ot2 and neutralize its antiviral activity. These results suggest that this region is located on the surface of the IFN molecule. Free peptides, or conjugates with bovine se-
TABLE 1 Location of synthetic peptides within the C-terminus of the human IFN a-2 molecule. Peptide code
Sequencea, b
2433 2438 2444 2944 2938 3444
(124)R-I-T-L-Y-L-K-E-K-K(133) (124)R-I-T-L-Y-L-K-E-K-K-Y-S-P-C-A(138) (124)R-I-T-L-Y-L-K-E-K-K-Y-S-P-C-A-W-E-V-V-R-A(144) (129)L-K-E-K-K-Y-S-P-C-A-W-E-V-V-R-A(144) (129)L-K-E-K-K-Y-S-P-C-A(138) (134)Y-S-P-C-A-W-E-V-V-R-A(144) apresented in one-letter code. bThe numbering of amino acid residues is reported according to [8].
16
rum albumin, did not display antiviral activity, nor could they inhibit the activity of IFN-ct2 [8]. According to our unpublished data, the IFN-ct2 peptides from the 124-144 C-terminal region do not influence the activity of NK cells, in contrast to the native IFN-a2 molecule. Growth inhibition of normal and tumor cells in vitro and in vivo is known to be one of the important biological characteristics of IFNs [18]. In the present study we have investigated the influence of previously synthesized peptides [8] on the proliferative response of human blood cells. Fig. 1A and B shows the antiproliferative activities of six synthetic peptides of the 124- 144 region of the human IFN a-2 molecule expressed on blood leukocytes in vitro. It was found that both IFN ct-2, and the peptide corresponding to the 124-138amino acid sequence of the human IFN a-2 molecule, inhibited proliferation of human T lympho-
A 30.
cytes in the presence of PHA (Fig. 1A) or IL-2 (Fig. 1B). Proliferation of mononuclear cells as well as separated T lymphocytes was also inhibited by IFN and peptide 2438 (data not shown). It was further shown that the cytotoxic effect of peptide 2438, at the concentrations used, was negligible as judged from the measurement of the proportion of dead cells in comparison to the control. The measurement was made by staining with trypan blue. Interestingly, the peptide 2444 which includes peptide 2438 was biologically inert (Fig. 1A and B). This finding could be attributed to conformational changes induced by additional amino acid residues. A similar action of peptide 2438 and IFN o~-2was not seen in the case of individual donor cells with abnormal sensitivity to IFN, both very high and very low (results not shown). Pretreatment of cells with PHA or IL-2, for 18- 24 h blocked the antiproliferative activity of both peptide 2438 and IFN (results not shown). The cytostatic effect of peptide 2438 and of IFN was dose-dependent (Figs. 2 and 3). Inhibition of
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........................................................... 0
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2444 2944 2438 2938 2433 3444
Fig. I. Proliferation of human blood T lymphocytes cultured with IFN ct-2,or synthetic pcptidcs, in the presence of P H A (A) or IL-2 (B). Control column (labeled "C") demonstrates proliferation of cellswith medium alone. Concentrations: P H A , 2 #g/ ml; IL-2, 20U/ml; IFNa-2, 10001U/ml; synthetic peptides, I0-6 M. pH]Thymidinc incorporation was tested in the last 15 h of 3 days of culture. S E M was calculated from 12 experiments.
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10-7 10-6 i0-5 concentration o! peptide 2438 (M) Fig. 2. Inhibition of PHA-induccd T lymphocyte proliferation by peptidc 2438 (dose-dependency curve). Broken lines: I, P H A alone; 2, medium alone. Concentrations: P H A , 2/~g/ml, pep-
tide 253910-5- 10-?M. SEM was calculatedfrom 4 experiments. 17
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.
.
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--
.
.
.
.
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Fig. 3. PHA (2 tzg/ml)-inducedproliferative response of human T lympbocytesin the presence of different IFNc¢-2concentrations: peptide 2438 10 -6 M; IFNc¢-2, 12.5- 500 IU/ml. Upper line (labeled "1"), IFNa-2 alone; lower line (labeled "2"), IFNc~-2plus peptide 2438. Columns: labeled "3", level of proliferative response in the presence of PHA (2 ~zg/ml)and peptide 2438 (10 6 M ) ; labeled "4", in the presence of PHP, only. SEM was calculated from 4 experiments.
the proliferative response to P H A was increased when peptide 2438 was added to the cell cultures simultaneously with IFN ~-2 (Fig. 3). Fig. 3 demonstrates the level of proliferative response of human T lymphocytes to P H A at various concentrations of IFN o~-2 alone (upper line), or in the presence of 10-6 M peptide 2438 (lower line). Simultaneous addition of both IFN and peptide 2438 provided a more powerful inhibitory effect than each agent taken separately. The results described above support three possibilities. (i) The 1 2 4 - 138 region of the human IFNc~2 molecule may contribute to the antiproliferative action of IFN, and peptide 2438 exerts its activity by the same pathway as IFN; (ii) peptide 2438 transfers antiproliferative activity by a distinct mechanism compared to IFN-~2; (iii) situations (i) and (ii) are realized together. Other results have shown that a synthetic peptide corresponding to the 1 3 1 - 138 C-terminal region of the IFN-o~2 molecule influences the proliferative 18
response of mouse thymocytes in vitro [19]. It is interesting that the effects exhibited by IFN-c~2 and the synthetic peptide in mouse and human systems are quite opposite. In a study employing ]25I-labeled synthetic peptide of the 131 - 133 region of human IFN-c¢2, it was demonstrated that the peptide has specific receptors on mouse thymocytes which are common to IFN-cz2 and thymosin-c~ [19]. Several functionally important epitopes on the human IFN-o~2 molecule have been located with the aid of mouse monoclonal antibodies: three individual and two partially overlapping antigenic determinants are responsible for various biological activities of IFN [20]. Study of the biological effects mediated by different recombinant and hybrid IFN molecules has allowed the identification of an IFN subtype displaying strong antiviral and antiproliferative activities, but lacking the ability to boost NK cytotoxicity. This suggests the existence of distinct functional domains of the IFN molecule responsible for different biological activities [21, 22]. Using site-directed mutagenesis of cloned IFN genes it was shown that the C-terminus played an important role in antiproliferative and antiviral activities of IFN [23], as well as in its binding with the receptor [11, 24]. The results reported above indicate that the biological effects of IFN can be dissociated, and involve different molecular mechanisms. This is supported by the experimental data suggesting that the biological activities of IFN are realized through distinct pathways [25, 26]; and involve different parts of the IFN molecule [20 - 24]. It would be interesting to test the hypothesis that different parts of the IFN molecule mediate distinct biological activities, and to study their individual contributions. References
[1] Henco,K., Brocies, J., Fujisawa, A., Fujisawa, J.-l., Haynes, J. R., Hochstadt, J., Kovacic,T., Pasek, M., Schambock, A., Schmid, J., Todokoro,K., Walchli, M., Nagata, S. and Weissman, C. (1985) J. Mol. Biol. 185,227. [2] Balkwill, F. R. and Burke, F. (1989) Immunol. Today 10, 299. [3] Streuli, M., Hall, A., Boll, W., Stewart, W. E., Nagata, S. and Weissman, C. (1981) Proc. Natl. Acad. Sci. USA 78, 2848.
[4] Shafferman, A., Velan, B., Cohen, S., Leitner, M. and Grosfeld, H. (1987) J. Biol. Chem. 262,6227. [5] Lydon, N. B., Farre, C., Bove, S., Neyret, O., Benuareau, S., Levine, A.M., Sulig, G. F., Nagabhushan, T. L. and Trotta, P. P. (1985) Biochemistry 24, 4131. [6] Sternberg, M. J.E. and Cohen, F.E. (1982) Int. J. Biol. Macromol. 4, 137. [7] Velan, B., Cohen, S., Grosfeld, H., Leitner, M. and Shafferman, A. (1985) J. Biol. Chem. 260, 5498. [8] Shevalier, A. F., Samukov, V. V., Ofitserov, V.I., Kalashnikov, V., Mizenko, G. A. and Kolokoltsov, A. A. (1990) Bioorg. Khim. USSR 16, 916. [9] Boyum, A. (1968) Stand. J. Clin. Invest. 21, 97. [lo] Aman, P., Ehlin-Henriksson, B. and Klein, G. (1984) J. Exp. Med. 159,208. [11] Weber, H., Valenzuela, D., Lujber, G., Gubler, M. and Weissman, C. (1987) EMBO J. 6, 591. [12] Taylor-Papadimitriou, J., Shearer, M. and Griffin, D. (1987) J. Immunol. 139, 3375. 1131 McInnes, B., Chambers, P. J., Cheetham, M. W., Beilharz, M. W. and Tymms, M. J. (1989) J. Interferon Res. 9, 305. [14] Fish, E.N., Banergee, K. and Stebbing, N. J. (1989) J. Interferon Res. 9, 97. [15] Arnheiter, H., Ohoro, M., Smith, M., Gutte, B. and Zoon, K. C. (1983) Proc. Natl. Acad. Sci. USA 80, 2539. [16] Ackerman, S. K., Zur Nedden, D., Heintzelman, M., Hunkapiller, M. and Zoon, K. (1984) Proc. Natl. Acad. Sci. USA 81. 1045.
[17] Leist, T. and Thomas, R. M. (1984) Biochemistry 23,254l. [18] Borden, E. C., Hogan, T. S. and Voelkel, J. G. (1982) Cancer Res. 42, 4948. [19] Zav’yalov, V. P., Navolotskaya, E. V., Abramov, V. M., Galactionov, V. G., Isaev, I. S., Kaurov, 0. A., Kozhich, A. T., Maiorov, A. V., Prusakov, A. N., Vasilenko, R. N. and Volodina, E. Yu. (1991) FEBS Lett. 278, 187. [20] Cebrian, M., Yague, E., De Landazuri, M. 0.. RodiguezMoya, M., Fresno, M., Pezzi, N., Uamazares, S. and Sanchez-Madrid, F. (1987) J. Immunol. 138, 484. [21] Ortaldo, J. R., Mason, A., Rehberg, E., Moschera, J., Kelder, B., Pestka, S. and Herberman, R. B. (1983) J. Biol. Chem. 258, 1501 I. [22] Ortaldo, J. R., Herberman, R. B., Harvey, C., Osheroff, P., Pan, Y-C. E., Kelder, B. and Pestka, S. (1984) Proc. Natl. Acad. Sci. USA 81, 4926. [23] Tymms, M. J., McInnes, B., Alin, P., Linnane, A. W. and Cheetham, B. F. (1990) Genet. Anal. Tech. Appl. 7, 53. 1241 Cheetham, B. F., McInnes, B., Mantamadiotis, T., Murray, P. J., Alin, P., Bourke, P., Linnane, A. W. and Tymms, M. J. (1991) Antiviral Res. 15, 27. [25] Forti, R. L., Mitchell, W.M., Hubbard, W. C., Workman, R. J. and Forbes, J. T. (1984) Proc. Natl. Acad. Sci. USA 81, 170. 1261 Kohn, L. D., Fridman, R. M., Holmes, J. M. and Lee, G. (1976) Proc. Natl. Acad. Sci. USA 73, 3695.
19
Synthetic peptide with antiproliferative activity: a short C-terminal fragment of the human interferon o -2 molecule A. V. D a n i l k o v i c h l, K. V. F r e z e 1, A. F. Shevalier 2, V. V. S a m u k o v 3, A. F. K i r k i n I a n d M. V. Gusev l 1Department of Cellular Physiology and Immunology, Biological Faculty, Moscow State University, Moscow; 2M. M. Shemyakin Institute of Bioorganic Chemistry, Moscow, and 3All Union Research Institute of Molecular Biology, Koltsovo, Novosibirsk Region, U.S.S.R. (Received 25 January 1991; revision received 23 July 1991; accepted 25 July 1991)
1. Summary The biological activity of six synthetic peptides of the 124 - 144 region of the human interferon a-2 (IFN or-2) molecule was studied. Peptides were examined for their ability to inhibit mitogen induced proliferation of human blood cells in vitro. Only the peptide corresponding to the amino acid sequence 1 2 4 - 1 3 8 (2438) possessed IFN-like antiproliferative activity. Other tested synthetic peptides did not affect cell proliferation in this experimental system. As with the native IFN or-2 molecule, the inhibitory effect of the peptide 2438 was dose-dependent. On simultaneous addition of peptide 2438, antiproliferative activity of IFN or-2 was enhanced. Direct cytotoxic effects of synthetic peptide 2438 were not revealed. These results suggest that a synthetic peptide corresponding to the 1 2 4 - 138-amino acid sequence of the human IFN o~-2 molecule serves as a cytostatic agent.
2. Introduction H u m a n o~-IFNs are a family of proteins which includes numerous subtypes showing over 75% homology of amino acid sequences [1]. IFN-a exerts various biological activities in different cell types, Key words: IFN a-2; Synthetic peptide; Human; T lymphocyte; Proliferation Correspondence to: Alia V. Danilkovich, Department of Cellular Physiology and Immunology, Biological Faculty, Moscow State University, Lenin's Hill, Moscow 119899, U.S.S.R.
both in vivo and in vitro [2]. Streuli et al. [3] have postulated that the IFN-a molecule contains at least two epitopes which interact with the receptor, one of them being located in the N-terminus and the other in the C-terminus of the IFN-o~ molecule. It was shown that the N-terminal part of the IFN-a molecule manifests antiviral and antiproliferative activities, and binds to the cellular receptor as does the whole IFN molecule [4]. At present there are no experimental data unambiguously confirming the existence of C-terminal active sites similar to those located in the Nterminus of the IFN c~-2 molecule. Other data support the involvement of the C-terminus in biological functions of IFN: (i) the Cys138 amino acid residue of the human IFN a-2 molecule is known to form the S - S bond important for biological activity of IFN [5]; (ii) a model of human IFN-a tertiary structure predicts that the fragment 1 2 0 - 147 is located near the N-terminal active site of IFN [6]; (iii) the amino acid sequence 1 2 0 - 1 4 7 of the IFN-ot molecule is highly (more than 86%) homologous among all the members of the family [7]. In the present study, we used previously synthesized peptides of the 1 2 4 - 144 segment of the IFN a-2 molecule [8] and examined their effects on the cell blast transformation response induced by polyclonal mitogens such as phytohemagglutinin (PHA) and interleukin-2 (IL-2) in vitro. The results demonstrate that like the intact IFN molecule, peptide 2438 corresponding to the 1 2 4 - 138-amino acid sequence of human IFN or-2 molecule can inhibit proliferation of mitogen-stim-
0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
15
ulated blood leukocytes in vitro. 3. Materials and Methods
3.1. Synthetic peptides A list of synthetic peptides used in these experiments and their location within the COOH-terminal part of the human IFN a-2 molecule is shown in Table 1. Their synthesis was performed as described [8].
3.2. Separation of lymphocytes Mononuclear cells were separated from the blood of healthy donors according to the method of Boyum [9]. The adherent cells were removed by incubation on plastic Petri dishes for 1 h at 37 °C in an atmosphere of 5°7o CO 2. The suspension of lymphocytes was divided into adherent and non-adherent subsets on a nylon wool column [10]. The nonadherent fraction was eluted with RPMI-1640 medium supplemented with 5°7o heat-inactivated fetal calf serum. This fraction contains T lymphocytes and null cells with small amounts of monocytes and B lymphocytes. 3.3. Proliferation assay Mononuclear cells, or separated T lymphocytes, were cultured in RPMI-1640 medium (Sigma) supplemented with 5°70 heat-inactivated fetal calf serum (Sigma), 100mM L-glutamine, 50/~g/ml gentamycin and 10/~M 2-mercaptoethanol in a humid atmosphere containing 5°70 CO 2 at 37 °C. 200/~l of lymphocyte suspension (mononuclear
cells or separated T-lymphocytes), at a concentration of 106 cells/ml in a 96-well flat-bottomed plate, were cultured in media containing P H A (2/~g/ml) or IL-2 (20 U/ml) in the presence of IFN o~-2 or synthetic peptides or IFN plus peptide for 75h. For the last 15h of culture, [3H]thymidine (0.5 tzCi/well; Amersham Corporation) was added. Cells were harvested and their radioactivity measured in a liquid scintillation counter (Rack-Beta LKB-Wallac, Sweden). P H A was obtained from Serva Laboratories. H u m a n recombinant IFN a-2 and human recombinant IL-2 were kindly provided by Dr. G. Chipens from the Institute of Organic Synthesis, Riga, Latvia, U.S.S.R. 4. Results and Discussion
Numerous experimental results emphasize the importance of the COOH-terminal part of the IFNot molecule in its biological action [11 - 14]. Earlier, six peptides of the 1 2 4 - 144 region of the human IFN a-2 molecule were synthesized (Table 1), and their antiviral potential was examined. It was found that, in contrast to the native IFN molecule, these peptides did not reveal antiviral activity [8]. This result agrees with the data on antiviral activity of different peptides from the COOHterminus of IFN-et molecules [15 - 17]. Interestingly, rabbit antisera developed against peptides 1 2 4 - 138 and 1 2 9 - 144 showed an ability to bind recombinant IFN-ot2 and neutralize its antiviral activity. These results suggest that this region is located on the surface of the IFN molecule. Free peptides, or conjugates with bovine se-
TABLE 1 Location of synthetic peptides within the C-terminus of the human IFN a-2 molecule. Peptide code
Sequencea, b
2433 2438 2444 2944 2938 3444
(124)R-I-T-L-Y-L-K-E-K-K(133) (124)R-I-T-L-Y-L-K-E-K-K-Y-S-P-C-A(138) (124)R-I-T-L-Y-L-K-E-K-K-Y-S-P-C-A-W-E-V-V-R-A(144) (129)L-K-E-K-K-Y-S-P-C-A-W-E-V-V-R-A(144) (129)L-K-E-K-K-Y-S-P-C-A(138) (134)Y-S-P-C-A-W-E-V-V-R-A(144) apresented in one-letter code. bThe numbering of amino acid residues is reported according to [8].
16
rum albumin, did not display antiviral activity, nor could they inhibit the activity of IFN-ct2 [8]. According to our unpublished data, the IFN-ct2 peptides from the 124-144 C-terminal region do not influence the activity of NK cells, in contrast to the native IFN-a2 molecule. Growth inhibition of normal and tumor cells in vitro and in vivo is known to be one of the important biological characteristics of IFNs [18]. In the present study we have investigated the influence of previously synthesized peptides [8] on the proliferative response of human blood cells. Fig. 1A and B shows the antiproliferative activities of six synthetic peptides of the 124- 144 region of the human IFN a-2 molecule expressed on blood leukocytes in vitro. It was found that both IFN ct-2, and the peptide corresponding to the 124-138amino acid sequence of the human IFN a-2 molecule, inhibited proliferation of human T lympho-
A 30.
cytes in the presence of PHA (Fig. 1A) or IL-2 (Fig. 1B). Proliferation of mononuclear cells as well as separated T lymphocytes was also inhibited by IFN and peptide 2438 (data not shown). It was further shown that the cytotoxic effect of peptide 2438, at the concentrations used, was negligible as judged from the measurement of the proportion of dead cells in comparison to the control. The measurement was made by staining with trypan blue. Interestingly, the peptide 2444 which includes peptide 2438 was biologically inert (Fig. 1A and B). This finding could be attributed to conformational changes induced by additional amino acid residues. A similar action of peptide 2438 and IFN o~-2was not seen in the case of individual donor cells with abnormal sensitivity to IFN, both very high and very low (results not shown). Pretreatment of cells with PHA or IL-2, for 18- 24 h blocked the antiproliferative activity of both peptide 2438 and IFN (results not shown). The cytostatic effect of peptide 2438 and of IFN was dose-dependent (Figs. 2 and 3). Inhibition of
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.......
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........................................................... 0
0
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2444 2944 2438 2938 2433 3444
Fig. I. Proliferation of human blood T lymphocytes cultured with IFN ct-2,or synthetic pcptidcs, in the presence of P H A (A) or IL-2 (B). Control column (labeled "C") demonstrates proliferation of cellswith medium alone. Concentrations: P H A , 2 #g/ ml; IL-2, 20U/ml; IFNa-2, 10001U/ml; synthetic peptides, I0-6 M. pH]Thymidinc incorporation was tested in the last 15 h of 3 days of culture. S E M was calculated from 12 experiments.
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10-7 10-6 i0-5 concentration o! peptide 2438 (M) Fig. 2. Inhibition of PHA-induccd T lymphocyte proliferation by peptidc 2438 (dose-dependency curve). Broken lines: I, P H A alone; 2, medium alone. Concentrations: P H A , 2/~g/ml, pep-
tide 253910-5- 10-?M. SEM was calculatedfrom 4 experiments. 17
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.
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.
.
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Fig. 3. PHA (2 tzg/ml)-inducedproliferative response of human T lympbocytesin the presence of different IFNc¢-2concentrations: peptide 2438 10 -6 M; IFNc¢-2, 12.5- 500 IU/ml. Upper line (labeled "1"), IFNa-2 alone; lower line (labeled "2"), IFNc~-2plus peptide 2438. Columns: labeled "3", level of proliferative response in the presence of PHA (2 ~zg/ml)and peptide 2438 (10 6 M ) ; labeled "4", in the presence of PHP, only. SEM was calculated from 4 experiments.
the proliferative response to P H A was increased when peptide 2438 was added to the cell cultures simultaneously with IFN ~-2 (Fig. 3). Fig. 3 demonstrates the level of proliferative response of human T lymphocytes to P H A at various concentrations of IFN o~-2 alone (upper line), or in the presence of 10-6 M peptide 2438 (lower line). Simultaneous addition of both IFN and peptide 2438 provided a more powerful inhibitory effect than each agent taken separately. The results described above support three possibilities. (i) The 1 2 4 - 138 region of the human IFNc~2 molecule may contribute to the antiproliferative action of IFN, and peptide 2438 exerts its activity by the same pathway as IFN; (ii) peptide 2438 transfers antiproliferative activity by a distinct mechanism compared to IFN-~2; (iii) situations (i) and (ii) are realized together. Other results have shown that a synthetic peptide corresponding to the 1 3 1 - 138 C-terminal region of the IFN-o~2 molecule influences the proliferative 18
response of mouse thymocytes in vitro [19]. It is interesting that the effects exhibited by IFN-c~2 and the synthetic peptide in mouse and human systems are quite opposite. In a study employing ]25I-labeled synthetic peptide of the 131 - 133 region of human IFN-c¢2, it was demonstrated that the peptide has specific receptors on mouse thymocytes which are common to IFN-cz2 and thymosin-c~ [19]. Several functionally important epitopes on the human IFN-o~2 molecule have been located with the aid of mouse monoclonal antibodies: three individual and two partially overlapping antigenic determinants are responsible for various biological activities of IFN [20]. Study of the biological effects mediated by different recombinant and hybrid IFN molecules has allowed the identification of an IFN subtype displaying strong antiviral and antiproliferative activities, but lacking the ability to boost NK cytotoxicity. This suggests the existence of distinct functional domains of the IFN molecule responsible for different biological activities [21, 22]. Using site-directed mutagenesis of cloned IFN genes it was shown that the C-terminus played an important role in antiproliferative and antiviral activities of IFN [23], as well as in its binding with the receptor [11, 24]. The results reported above indicate that the biological effects of IFN can be dissociated, and involve different molecular mechanisms. This is supported by the experimental data suggesting that the biological activities of IFN are realized through distinct pathways [25, 26]; and involve different parts of the IFN molecule [20 - 24]. It would be interesting to test the hypothesis that different parts of the IFN molecule mediate distinct biological activities, and to study their individual contributions. References
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