immortalized by the temperature-sensitive large T antigen were successfully transplanted without tumor formation. We wish to thank Drs J. Heitzmann and M. Cohn for providing the cell lines, Dr C. Cepko for viral infection protocols, and A. M. Wesley for excellent technical assistance. Dr Bredesen is supported by National Institutes of Health Fellowship NS 08100 from National Institute of Neurological Disorders and Stroke and Drs Hisanaga and Sharp, by NS 24666 and the Veterans Administration Merit Review Program.
1. Perlow MJ, Freed WJ, Hoffer BJ, et al. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979;204:643-647 2. Bjorklund A, Dunnett SB, Stenevi U, et al. Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980;199:307-333 3. Madrazo I, Drucker-Colin R, Diaz V, et al. Open microsurgical autograft of adrenal medulla to the right caudate nucleus in two patients with intractable Parkinson’s disease. N Engl J Med 1987;316:831-834 4. Madrazo I, Leon V, Torres C, et al. Transplantation of fetal substantia nigra and adrenal medulla to the caudate nucleus in two patients with Parkinson’s disease. N Engl J Med 1988;318: 51 (Letter) 5. Backlund E-0, Granberg P-0, Hamberger B, et al. Transplantation of adrenal medullary tissue to striatum in parkinsonism. First clinical trials. J Neurosurg 1985;62:169-173 6. Goetz CG, Olanow CW, Koller WC, et al. Multicenter study of autologous adrenal medullary transplantation to the corpus striain patients with advanced Parkinson’s disease. N Engl J Med 1989;320:337-34 1 lovnt RT. Gash DM. Neural transplants: are we ready? Ann 8. Gage FH, Wolff JA, Rosenberg MB, et al. Grafting genetically modified cells to the brain: possibilities for the future. Neuroscience 1987;23:795-807 9. Bredesen DE, Hisanaga K, Sharp FR. Neural transplantation with temperature-sensitive immortalized neural cells. Neurol 1989;39(suppl 1):124 (Abstract) 10. Chen YC, Hayman MJ, Vogt PK. Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus. Cell 1977;11:513-521 11. Giotta GJ, Heitzmann J, Cohn M. Properties of two temperature-sensitive Rous sarcoma virus transformed cerebellar cell lines. Brain Res 1980;202:445-448 12. Greenberg ME, Brackenbury R, Edelman GM. Alteration of neural cell adhesion molecule (N-CAM) expression after neuronal cell transformation by Rous sarcoma virus. Proc Natl Acad Sci USA 1984;81:969-973 13. Bredesen DE, Scott MRD, Torchia T, Prusiner SB. Differentiation modulates cellular prion protein expression and targeting. Neurology 1989;39(suppl 1):396 (Abstract) 14. Price J, Turner D, Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci USA 1987;84:156-160 15. Whitremore SR, Holets VR, Gonzalez-Carvajal M, Levy D. Transplantation of a hippocampally-derived,immortal, temperature-sensitive, neuronal cell line into adult rat CNS. Neurosci Abstr 1988;233.1 (Abstract)
Interferon Beta Augments Suppressor Cell Function in Multiple Sclerosis Avertano Noronha, MBBS, MD, Angela Toscas, BA, and Mark A. Jensen, BS Suppressor cell function has been previously reported to be decreased in patients with progressive multiple sclerosis (MS). The abnormality could not be corrected in vitro and was present even after patients were treated with immunosuppressive agents. We now report that interferon beta augments suppressor function in vitro in progressive MS. Nonspecific suppressor cell function as measured in a concanavalin A (Con A) suppressor assay was reduced in 24 MS patients (mean percent suppression, 19.6 2.2) when compared to 19 normal subjects (mean percent suppression, 35.0 & 3.3). The data are highly significant ( p < 0.001). When recombinant human interferon beta (lo3unidml) was added to lymphocyte cultures with Con A, suppressor activity improved significantly. The mean percent suppression improved from 19.6 f 2.2 to 37.8 f 2.6 in MS ( p < 0.001) and from 35.0 2 3.3 to 46.2 f 3.5 ( p < 0.025) in control subjects. This study shows that recombinant interferon beta improves suppressor function in humans, an effect that is particularly significant in progressive MS. Noronha A, Toscas A, Jensen MA. Interferon beta augments suppressor cell function in multiple sclerosis. Ann Neurol 1990;27:207-210
Multiple sclerosis (MS) is a disease confined to the central nervous system; demyelination and inflammation in brain and the presence of oligoclonal bands and activated cells in cerebrospinal fluid are characteristic features [l-43. However, abnormalities of immune function are reported to occur in peripheral blood, raising the possibility that the disease may be immune mediated (reviewed in [S}). One such abnormality is an impairment of nonspecific suppressor cell function. Suppressor cell function as measured by the concanavalin A (Con A) suppressor assay and by pokeweed mitogen-induced immunoglobulin secretion is consistently abnormal in patients with progressive MS l6-81. The defect resides in T cells, specifically the CDS-positive subset. In addition, CD&positive T-cell lines from MS patients are shown to be functionally ~~~~~
~~
From the Department of Neurology and The Brain Research Institute, University of Chicago, Chicago, IL. Received Jun 7, 1989, and in revised form Aug 7. Accepted for publication Aug 10, 1989. Address correspondence to Dr Noronha, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637.
Copyright 0 1990 by the American Neurological Association 207
impaired in assays of suppressor cell function 19, lo}. The reason for the suppressor cell defect remains unknown. To date, the suppressor abnormality has not been corrected either in vivo with immunosuppressive agents or in vitro. We now report that human recombinant interferon beta (Betaseron, Triton BioSciences, Inc, Alameda, CA) can augment suppressor cell function in vitro, particularly in MS patients. Methods We studied 24 patients (mean age ? SD, 43.0 5 8.0 years) with clinically definite MS. All patients had severe progressive disease and had not received treatment with either corticosteroids or other immunosuppressive drugs for 1 year before the study. Nineteen healthy laboratory workers (mean age 2 SD, 28.0 2 6.9 years) served as control subjects. Since the purpose of this study was to examine the effect of interferon beta on suppressor function in MS, and not the specificity of the suppressor abnormality in MS, patients with other neurological diseases were not studied. Peripheral blood mononuclear cells (PBMs) were isolated on Ficoll-sodium diatritoate (Hypaque) gradients. Suppressor cells (S cells) were generated by culturing cells with Con A for 48 hours (1 x lo6 PBMslml in RPMI + 10% fetal bovine serum 20 mM MOPS + 10 mM gentamicin). Cells cultured in medium alone (C cells) served as a control. To test the effect of interferon beta, we added Betaseron, lo3 unitdml, to cultures containing Con A. (A doseresponse curve showed that Betaseron was optimally effective in augmenting suppressor activity of Con A-treated cells at lo3 unitdml. In comparison, patients with remittingrelapsing MS entered in a clinical trial are given 45 x lo6 units every 2-3 days.) After 48 hours, cells were washed and treated with mitomycin C (50 pdml). S or C cells, 1 x lo5, were added to 1 x lo5 freshly isolated responder cells from a normal donor (R cells) and Con A and cultured in quadruplicate in wells of a 96-well round-bottom microtiter plate. After 72 hours, cells were pulsed with 3H-thymidine for 5 hours and harvested in an automated harvester, and radioactivity was determined in a scintillation counter. Percent suppression was calculated as follows:
+
1-
cpm of cultures (lo5 R cells cpm of cultures (lo5 R cells
+ lo5 S cells + Con A) + lo5 C cells + Con A) x 100%
Data are expressed as mean percent suppression 2 SEM. Statistical analysis was performed with use of the Student’s t test.
Results Suppressor function was found to be abnormal in progressive MS. The mean percent suppression was 19.6 & 2.2 in patients with progressive MS, as compared to 35.0 -+ 3.3% in healthy control subjects. The difference is highly significant ( p < 0.001). These results confirm previous reports. We also found that suppressor function is augmented in vitro by recombinant interferon beta. The
208 Annals of Neurology
Vol 27 N o 2 February 1990
6ol 50
.-a
T
40
?! n 30
In
8
20
10
n
MS
CONTROLS
Effect of intwfwon beta on concanavalin A (Con A) suppressor activity. Data from the Con A suppressor assay (dark bars, Con A alone; shaded bars, Con A plus interjeron beta)for 24 patients with progressive MS and 19 healthy control subjects are expressed as mean percent suppression 2 SEM.
addition of Betaseron to Con A-treated cultures augumented suppressor cell function (Figure). The mean percent suppression improved from 19.6 +. 2.2 to 37.8 & 2.6% in MS. The difference is highly significant ( p < 0.001). A modest increase in suppressor function was also noted in control subjects, from 35.0 -+ 3.396 to 46.2 +. 3.5% ( p < 0.025).
Discussion This study shows for the first time that suppressor function can be improved in vitro in MS patients. It confirms previous observations that suppressor function is impaired in patients with progressive MS. Suppressor function was augmented by a human recombinant interferon beta (Betaseron). The effect, while most marked in MS patients with defective suppressor cell function, was also seen in normal subjects, indicating that interferon beta acts on suppressor cells in general. A considerable body of work shows that interferons modulate immunological function {l11. Immune responses can be either stimulated or depressed, depending on the type of interferon used. Type I1 interferon (interferon gamma) has been shown to augment the expression of class I1 major histocompatibility complex (MHC) molecules on peripheral blood cells. Class I1 MHC expression, which is low to absent in brain, can be induced by interferon gamma in cultured astrocytes but not in neurons. Once stimulated to express class I1 MHC molecules, astrocytes can act as antigen-presenting cells {12J Similarly, endothelial cells can be induced to express class I1 MHC molecules and present antigen {13, 141. Such an effect observed in vitro may contribute to inflammatory responses in vivo. In fact, interferon gamma has been reported to increase the number of exacerbations in MS C151.
In contrast, type I interferons (alpha, beta) downregulate class I1 MHC expression. Interferon beta prevents the up-regulation of class I1 molecules on macrophages, induced by interferon gamma {16, 17). Recently, type I interferons (alpha, beta) have been reported to be beneficial in decreasing the number of attacks in remitting-relapsing MS 118, 191. In contrast, another study showed that relapses were fewer in both patients and control subjects when recombinant interferon alfa-2 was used E201. Our in vitro studies using recombinant interferon beta were performed with blood from patients with progressive disease. Immunological effects seen in vitro may not necessarily occur in vivo. It would be of interest to know if improvement in in vitro suppressor function and clinical improvement occur in patients with progressive MS who are treated with Betaseron. Such a study is planned. In addition to its effect on peripheral blood cells, interferon beta may act to down-regulate class I1 MHC expression in brain in demyelinating disease. We are currently evaluating this effect in experimental animals. Type I interferons are suppressive for several immune responses, including antibody production, delayed-type hypersensitivity, and graft rejection {2 1, 22). In one study, suppressor activity induced by Con A was largely attributed to type I interferon; it could be blocked by anti-interferon serum [23}. Interferon beta activates suppressor cells in mice and interferon alfa augments suppressor function in humans [24, 251. The precise manner in which interferon beta augments suppressor function remains to be determined. Possible explanations include effects on macrophages and on T cells. When type I interferon is added to macrophages, it down-regulates class I1 MHC expression induced by type I1 interferons on these cells {l6, 171. Interferon beta may therefore reduce the ability of macrophages to interact with primed T cells, thereby augmenting suppression. The other possibility is a direct effect of interferon beta on T cells. Interferon beta is reported to increase the expression of Ly-6 surface antigens on T cells and augment activation through the Ly-6 pathway 1261. We are presently studying the effect of interferon beta on isolated human CD8 clones. The augmentation of Con A suppressor activity by interferon beta is not due to further activation of PBMs; the degree of proliferation of PBMs after activation by Con A or interferon beta plus Con A was similar in MS patients and control subjects. In our assay system, interferon beta was effective when added simultaneously with Con A. Therefore, interferon beta may be providing a second amplification signal which may be transduced by putative second messengers such as diacylglycerol. In summary, our study provides evidence for a role
of interferon beta in suppressor function, an effect that is particularly noteworthy in MS.
~
~~
Supported by grants from the National Institutes of Health (1PO1NS-24575) and from Triton BioSciences, Inc. We thank Dr Barry Arnason, to whom we are continually indebted for his guidance.
References 1. Adams CWM. Pathology of multiple sclerosis: progression of the lesion. Br Med Bull 1977;33:15-20 2. Link H. Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid: chemical and immunological characterization with special reference to multiple sclerosis. Acta Neurol Scand 1967;43(suppl 28):l-136 3. Noronha A, Richman DP, Arnason BGW. Detection of in vivo stimulated cerebrospinal fluid lymphocytes by flow cytometry in patients with multiple sclerosis. N Engl J Med 1980;303:713717 4. HaIler DA, Fox DA, Manning ME, et al. In vivo activated T lymphocytes in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. N Engl J Med 1985;312:14051411 5 . Reder AT, Arnason BGW. Immunology of multiple sclerosis. In: Vinken PJ, Bruyn GW, Klawans HL, Koetsier JC, eds. Handbook of clinical neurology. Amsterdam: Elsevier, 1985: 337-395 6. Arnason BGW, Antel JP. Suppressor cell function in multiple sclerosis. Ann Immunol (Paris) 1978;129C:159-170 7. Antel JP, Brown M, Nicholas MK, et al. Activated suppressor cell dysfunction in progressive multiple sclerosis. J Neuroimmunol 1988;17:323-330 8. Oger J, Kastrukoff L, OGorman M, Paty DW. Progressive multiple sclerosis: abnormal immune functions in vitro and aberrant correlation with enumeration of lymphocyte subpopulations. J Neuroimmunol 1986;12:37-48 9. Antel JP, Noronha A. Correlation of lymphocyte phenotypes and immune function in multiple sclerosis (MS). In: Lowenthal A, Raus J, eds. Cellular and humoral immunological components of cerebrospinal fluid in multiple sclerosis. New York: Plenum, 1987:219-227 10. Antel JP, Bania M, Noronha A. Defective suppressor cell function mediated by T8+ cell lines from patients with progressive multiple sclerosis.J Immunol 1986;137:3436-3439 11. Friedman RM, Vogel SN. Interferons with special emphasis on the immune system. Adv Immunol 1983;34:97-133 12. Fontana A, Fierz W, Wekerle H. Astrocytes present myelin basic protein to encephalitogenic T-cell lines. Nature 1984; 307:273-276 13. Pober JS, Gimbrone MA, Cotran RS. Ia expression by vascular endothelium is inducible by activated T-cells and by human yinterferon. J Exp Med 1983;157:1339-1353 14. McCarron RM, Spatz M, Kempski 0,et al. Interaction between myelin basic protein-sensitized T lymphocytes and murine cerebral vascular endothelial cells. J Immunol 1986;137:34283435 15. Panitch H, Hirsch RL,Haley AS, Johnson KP. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987;1:893-895 16. Ling PD, Warren MK, Vogel SN. Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia
Brief Communication: Noronha et
al:
Interferon Beta in MS 209
antigen in murine macrophages. J Immunol 1985;135:18571863 17. Inaba K, Kitaura M, Kato T, et al. Contrasting effect of dp and y-interferons on expression of macrophage Ia antigens. J Exp Med 1986;163:1030-1035 18. Knobler RL, Panitch HS, Braheny SL. Systemic alpha-interferon therapy of multiple sclerosis. Neurology 1984;34:12731279 19. Jacobs L, Salazar AM, Herndon R. Intrathecally administered natural human fibroblast interferon reduces exacerbations of multiple sclerosis. Arch Neurol 1987;44:589-595 20. Camenga DL, Johnson KP, Alter M, et al. Systemic recombinant a-2 interferon therapy in relapsing multiple sclerosis. Arch Neurol 1988;43:1239- 1246 21. De Maeyer E, D e Maeyer-Guignard J, Vandeputte M. Inhi-
210 Annals of Neurology Vol 27 No 2 February 1990
22. 23. 24.
25. 26.
bition by interferon of delayed-type hypersensitivity in the mouse. Proc Natl Acad Sci USA 1975;72:1753-1757 Sonnenfeld G. Modulation of immunity by interferon. Lymphokine Res 1980;1:113-131 Kadish AS, Tansey FA, Yu GSM. Interferon as a mediator of human lymphocyte suppression. J Exp Med 1980;151:637-650 Schnaper HW, Aune TM, Pierce CW. Suppressor T cell activation by human leukocyte interferon. J Immunol 1983;131: 2 30 1-2 306 Aune TM, Pierce W. Activation of suppressor T-cell pathway by interferon. Proc Natl Acad Sci USA 1982;79:3808-3812 Dumont FJ. Treatment of resting T lymphocytes with interferon-dp augments their proliferative response to activation signals delivered through their surface Ly-6 antigen. Cell Immunol 1986;101:625-632
1. Perlow MJ, Freed WJ, Hoffer BJ, et al. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979;204:643-647 2. Bjorklund A, Dunnett SB, Stenevi U, et al. Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing. Brain Res 1980;199:307-333 3. Madrazo I, Drucker-Colin R, Diaz V, et al. Open microsurgical autograft of adrenal medulla to the right caudate nucleus in two patients with intractable Parkinson’s disease. N Engl J Med 1987;316:831-834 4. Madrazo I, Leon V, Torres C, et al. Transplantation of fetal substantia nigra and adrenal medulla to the caudate nucleus in two patients with Parkinson’s disease. N Engl J Med 1988;318: 51 (Letter) 5. Backlund E-0, Granberg P-0, Hamberger B, et al. Transplantation of adrenal medullary tissue to striatum in parkinsonism. First clinical trials. J Neurosurg 1985;62:169-173 6. Goetz CG, Olanow CW, Koller WC, et al. Multicenter study of autologous adrenal medullary transplantation to the corpus striain patients with advanced Parkinson’s disease. N Engl J Med 1989;320:337-34 1 lovnt RT. Gash DM. Neural transplants: are we ready? Ann 8. Gage FH, Wolff JA, Rosenberg MB, et al. Grafting genetically modified cells to the brain: possibilities for the future. Neuroscience 1987;23:795-807 9. Bredesen DE, Hisanaga K, Sharp FR. Neural transplantation with temperature-sensitive immortalized neural cells. Neurol 1989;39(suppl 1):124 (Abstract) 10. Chen YC, Hayman MJ, Vogt PK. Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus. Cell 1977;11:513-521 11. Giotta GJ, Heitzmann J, Cohn M. Properties of two temperature-sensitive Rous sarcoma virus transformed cerebellar cell lines. Brain Res 1980;202:445-448 12. Greenberg ME, Brackenbury R, Edelman GM. Alteration of neural cell adhesion molecule (N-CAM) expression after neuronal cell transformation by Rous sarcoma virus. Proc Natl Acad Sci USA 1984;81:969-973 13. Bredesen DE, Scott MRD, Torchia T, Prusiner SB. Differentiation modulates cellular prion protein expression and targeting. Neurology 1989;39(suppl 1):396 (Abstract) 14. Price J, Turner D, Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci USA 1987;84:156-160 15. Whitremore SR, Holets VR, Gonzalez-Carvajal M, Levy D. Transplantation of a hippocampally-derived,immortal, temperature-sensitive, neuronal cell line into adult rat CNS. Neurosci Abstr 1988;233.1 (Abstract)
Interferon Beta Augments Suppressor Cell Function in Multiple Sclerosis Avertano Noronha, MBBS, MD, Angela Toscas, BA, and Mark A. Jensen, BS Suppressor cell function has been previously reported to be decreased in patients with progressive multiple sclerosis (MS). The abnormality could not be corrected in vitro and was present even after patients were treated with immunosuppressive agents. We now report that interferon beta augments suppressor function in vitro in progressive MS. Nonspecific suppressor cell function as measured in a concanavalin A (Con A) suppressor assay was reduced in 24 MS patients (mean percent suppression, 19.6 2.2) when compared to 19 normal subjects (mean percent suppression, 35.0 & 3.3). The data are highly significant ( p < 0.001). When recombinant human interferon beta (lo3unidml) was added to lymphocyte cultures with Con A, suppressor activity improved significantly. The mean percent suppression improved from 19.6 f 2.2 to 37.8 f 2.6 in MS ( p < 0.001) and from 35.0 2 3.3 to 46.2 f 3.5 ( p < 0.025) in control subjects. This study shows that recombinant interferon beta improves suppressor function in humans, an effect that is particularly significant in progressive MS. Noronha A, Toscas A, Jensen MA. Interferon beta augments suppressor cell function in multiple sclerosis. Ann Neurol 1990;27:207-210
Multiple sclerosis (MS) is a disease confined to the central nervous system; demyelination and inflammation in brain and the presence of oligoclonal bands and activated cells in cerebrospinal fluid are characteristic features [l-43. However, abnormalities of immune function are reported to occur in peripheral blood, raising the possibility that the disease may be immune mediated (reviewed in [S}). One such abnormality is an impairment of nonspecific suppressor cell function. Suppressor cell function as measured by the concanavalin A (Con A) suppressor assay and by pokeweed mitogen-induced immunoglobulin secretion is consistently abnormal in patients with progressive MS l6-81. The defect resides in T cells, specifically the CDS-positive subset. In addition, CD&positive T-cell lines from MS patients are shown to be functionally ~~~~~
~~
From the Department of Neurology and The Brain Research Institute, University of Chicago, Chicago, IL. Received Jun 7, 1989, and in revised form Aug 7. Accepted for publication Aug 10, 1989. Address correspondence to Dr Noronha, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637.
Copyright 0 1990 by the American Neurological Association 207
impaired in assays of suppressor cell function 19, lo}. The reason for the suppressor cell defect remains unknown. To date, the suppressor abnormality has not been corrected either in vivo with immunosuppressive agents or in vitro. We now report that human recombinant interferon beta (Betaseron, Triton BioSciences, Inc, Alameda, CA) can augment suppressor cell function in vitro, particularly in MS patients. Methods We studied 24 patients (mean age ? SD, 43.0 5 8.0 years) with clinically definite MS. All patients had severe progressive disease and had not received treatment with either corticosteroids or other immunosuppressive drugs for 1 year before the study. Nineteen healthy laboratory workers (mean age 2 SD, 28.0 2 6.9 years) served as control subjects. Since the purpose of this study was to examine the effect of interferon beta on suppressor function in MS, and not the specificity of the suppressor abnormality in MS, patients with other neurological diseases were not studied. Peripheral blood mononuclear cells (PBMs) were isolated on Ficoll-sodium diatritoate (Hypaque) gradients. Suppressor cells (S cells) were generated by culturing cells with Con A for 48 hours (1 x lo6 PBMslml in RPMI + 10% fetal bovine serum 20 mM MOPS + 10 mM gentamicin). Cells cultured in medium alone (C cells) served as a control. To test the effect of interferon beta, we added Betaseron, lo3 unitdml, to cultures containing Con A. (A doseresponse curve showed that Betaseron was optimally effective in augmenting suppressor activity of Con A-treated cells at lo3 unitdml. In comparison, patients with remittingrelapsing MS entered in a clinical trial are given 45 x lo6 units every 2-3 days.) After 48 hours, cells were washed and treated with mitomycin C (50 pdml). S or C cells, 1 x lo5, were added to 1 x lo5 freshly isolated responder cells from a normal donor (R cells) and Con A and cultured in quadruplicate in wells of a 96-well round-bottom microtiter plate. After 72 hours, cells were pulsed with 3H-thymidine for 5 hours and harvested in an automated harvester, and radioactivity was determined in a scintillation counter. Percent suppression was calculated as follows:
+
1-
cpm of cultures (lo5 R cells cpm of cultures (lo5 R cells
+ lo5 S cells + Con A) + lo5 C cells + Con A) x 100%
Data are expressed as mean percent suppression 2 SEM. Statistical analysis was performed with use of the Student’s t test.
Results Suppressor function was found to be abnormal in progressive MS. The mean percent suppression was 19.6 & 2.2 in patients with progressive MS, as compared to 35.0 -+ 3.3% in healthy control subjects. The difference is highly significant ( p < 0.001). These results confirm previous reports. We also found that suppressor function is augmented in vitro by recombinant interferon beta. The
208 Annals of Neurology
Vol 27 N o 2 February 1990
6ol 50
.-a
T
40
?! n 30
In
8
20
10
n
MS
CONTROLS
Effect of intwfwon beta on concanavalin A (Con A) suppressor activity. Data from the Con A suppressor assay (dark bars, Con A alone; shaded bars, Con A plus interjeron beta)for 24 patients with progressive MS and 19 healthy control subjects are expressed as mean percent suppression 2 SEM.
addition of Betaseron to Con A-treated cultures augumented suppressor cell function (Figure). The mean percent suppression improved from 19.6 +. 2.2 to 37.8 & 2.6% in MS. The difference is highly significant ( p < 0.001). A modest increase in suppressor function was also noted in control subjects, from 35.0 -+ 3.396 to 46.2 +. 3.5% ( p < 0.025).
Discussion This study shows for the first time that suppressor function can be improved in vitro in MS patients. It confirms previous observations that suppressor function is impaired in patients with progressive MS. Suppressor function was augmented by a human recombinant interferon beta (Betaseron). The effect, while most marked in MS patients with defective suppressor cell function, was also seen in normal subjects, indicating that interferon beta acts on suppressor cells in general. A considerable body of work shows that interferons modulate immunological function {l11. Immune responses can be either stimulated or depressed, depending on the type of interferon used. Type I1 interferon (interferon gamma) has been shown to augment the expression of class I1 major histocompatibility complex (MHC) molecules on peripheral blood cells. Class I1 MHC expression, which is low to absent in brain, can be induced by interferon gamma in cultured astrocytes but not in neurons. Once stimulated to express class I1 MHC molecules, astrocytes can act as antigen-presenting cells {12J Similarly, endothelial cells can be induced to express class I1 MHC molecules and present antigen {13, 141. Such an effect observed in vitro may contribute to inflammatory responses in vivo. In fact, interferon gamma has been reported to increase the number of exacerbations in MS C151.
In contrast, type I interferons (alpha, beta) downregulate class I1 MHC expression. Interferon beta prevents the up-regulation of class I1 molecules on macrophages, induced by interferon gamma {16, 17). Recently, type I interferons (alpha, beta) have been reported to be beneficial in decreasing the number of attacks in remitting-relapsing MS 118, 191. In contrast, another study showed that relapses were fewer in both patients and control subjects when recombinant interferon alfa-2 was used E201. Our in vitro studies using recombinant interferon beta were performed with blood from patients with progressive disease. Immunological effects seen in vitro may not necessarily occur in vivo. It would be of interest to know if improvement in in vitro suppressor function and clinical improvement occur in patients with progressive MS who are treated with Betaseron. Such a study is planned. In addition to its effect on peripheral blood cells, interferon beta may act to down-regulate class I1 MHC expression in brain in demyelinating disease. We are currently evaluating this effect in experimental animals. Type I interferons are suppressive for several immune responses, including antibody production, delayed-type hypersensitivity, and graft rejection {2 1, 22). In one study, suppressor activity induced by Con A was largely attributed to type I interferon; it could be blocked by anti-interferon serum [23}. Interferon beta activates suppressor cells in mice and interferon alfa augments suppressor function in humans [24, 251. The precise manner in which interferon beta augments suppressor function remains to be determined. Possible explanations include effects on macrophages and on T cells. When type I interferon is added to macrophages, it down-regulates class I1 MHC expression induced by type I1 interferons on these cells {l6, 171. Interferon beta may therefore reduce the ability of macrophages to interact with primed T cells, thereby augmenting suppression. The other possibility is a direct effect of interferon beta on T cells. Interferon beta is reported to increase the expression of Ly-6 surface antigens on T cells and augment activation through the Ly-6 pathway 1261. We are presently studying the effect of interferon beta on isolated human CD8 clones. The augmentation of Con A suppressor activity by interferon beta is not due to further activation of PBMs; the degree of proliferation of PBMs after activation by Con A or interferon beta plus Con A was similar in MS patients and control subjects. In our assay system, interferon beta was effective when added simultaneously with Con A. Therefore, interferon beta may be providing a second amplification signal which may be transduced by putative second messengers such as diacylglycerol. In summary, our study provides evidence for a role
of interferon beta in suppressor function, an effect that is particularly noteworthy in MS.
~
~~
Supported by grants from the National Institutes of Health (1PO1NS-24575) and from Triton BioSciences, Inc. We thank Dr Barry Arnason, to whom we are continually indebted for his guidance.
References 1. Adams CWM. Pathology of multiple sclerosis: progression of the lesion. Br Med Bull 1977;33:15-20 2. Link H. Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid: chemical and immunological characterization with special reference to multiple sclerosis. Acta Neurol Scand 1967;43(suppl 28):l-136 3. Noronha A, Richman DP, Arnason BGW. Detection of in vivo stimulated cerebrospinal fluid lymphocytes by flow cytometry in patients with multiple sclerosis. N Engl J Med 1980;303:713717 4. HaIler DA, Fox DA, Manning ME, et al. In vivo activated T lymphocytes in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. N Engl J Med 1985;312:14051411 5 . Reder AT, Arnason BGW. Immunology of multiple sclerosis. In: Vinken PJ, Bruyn GW, Klawans HL, Koetsier JC, eds. Handbook of clinical neurology. Amsterdam: Elsevier, 1985: 337-395 6. Arnason BGW, Antel JP. Suppressor cell function in multiple sclerosis. Ann Immunol (Paris) 1978;129C:159-170 7. Antel JP, Brown M, Nicholas MK, et al. Activated suppressor cell dysfunction in progressive multiple sclerosis. J Neuroimmunol 1988;17:323-330 8. Oger J, Kastrukoff L, OGorman M, Paty DW. Progressive multiple sclerosis: abnormal immune functions in vitro and aberrant correlation with enumeration of lymphocyte subpopulations. J Neuroimmunol 1986;12:37-48 9. Antel JP, Noronha A. Correlation of lymphocyte phenotypes and immune function in multiple sclerosis (MS). In: Lowenthal A, Raus J, eds. Cellular and humoral immunological components of cerebrospinal fluid in multiple sclerosis. New York: Plenum, 1987:219-227 10. Antel JP, Bania M, Noronha A. Defective suppressor cell function mediated by T8+ cell lines from patients with progressive multiple sclerosis.J Immunol 1986;137:3436-3439 11. Friedman RM, Vogel SN. Interferons with special emphasis on the immune system. Adv Immunol 1983;34:97-133 12. Fontana A, Fierz W, Wekerle H. Astrocytes present myelin basic protein to encephalitogenic T-cell lines. Nature 1984; 307:273-276 13. Pober JS, Gimbrone MA, Cotran RS. Ia expression by vascular endothelium is inducible by activated T-cells and by human yinterferon. J Exp Med 1983;157:1339-1353 14. McCarron RM, Spatz M, Kempski 0,et al. Interaction between myelin basic protein-sensitized T lymphocytes and murine cerebral vascular endothelial cells. J Immunol 1986;137:34283435 15. Panitch H, Hirsch RL,Haley AS, Johnson KP. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987;1:893-895 16. Ling PD, Warren MK, Vogel SN. Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia
Brief Communication: Noronha et
al:
Interferon Beta in MS 209
antigen in murine macrophages. J Immunol 1985;135:18571863 17. Inaba K, Kitaura M, Kato T, et al. Contrasting effect of dp and y-interferons on expression of macrophage Ia antigens. J Exp Med 1986;163:1030-1035 18. Knobler RL, Panitch HS, Braheny SL. Systemic alpha-interferon therapy of multiple sclerosis. Neurology 1984;34:12731279 19. Jacobs L, Salazar AM, Herndon R. Intrathecally administered natural human fibroblast interferon reduces exacerbations of multiple sclerosis. Arch Neurol 1987;44:589-595 20. Camenga DL, Johnson KP, Alter M, et al. Systemic recombinant a-2 interferon therapy in relapsing multiple sclerosis. Arch Neurol 1988;43:1239- 1246 21. De Maeyer E, D e Maeyer-Guignard J, Vandeputte M. Inhi-
210 Annals of Neurology Vol 27 No 2 February 1990
22. 23. 24.
25. 26.
bition by interferon of delayed-type hypersensitivity in the mouse. Proc Natl Acad Sci USA 1975;72:1753-1757 Sonnenfeld G. Modulation of immunity by interferon. Lymphokine Res 1980;1:113-131 Kadish AS, Tansey FA, Yu GSM. Interferon as a mediator of human lymphocyte suppression. J Exp Med 1980;151:637-650 Schnaper HW, Aune TM, Pierce CW. Suppressor T cell activation by human leukocyte interferon. J Immunol 1983;131: 2 30 1-2 306 Aune TM, Pierce W. Activation of suppressor T-cell pathway by interferon. Proc Natl Acad Sci USA 1982;79:3808-3812 Dumont FJ. Treatment of resting T lymphocytes with interferon-dp augments their proliferative response to activation signals delivered through their surface Ly-6 antigen. Cell Immunol 1986;101:625-632
