- Email: [email protected]
Does cyclosporine act in vivo as it does in vitro?
Immunology Today, voL 7, No. 4, 1986
A regulatory Id/anti-ld network in a real mouse suffering from the illusion of the 'completeness axiom' can only consist of a single pair of complementary and highly degenerate 'blobs'. Experimentalists who have been inspired by the idea of an idiotype network can claim to be dealing with real mice, and they may well point to repeated demonstrations that Ids and anti-lds can exert strong effects on the immune response. Aside from a few examples of experiments that have not been generalizable, there is no body of data that cannot be adequately (often better) explained by the Associative Recognition Theory 7. The effects of immunogenic complexes made up of self (S) and foreign (F) components (i.e., S-F) formed in vivo, have been thoroughly analysed within the framework of the Associative Recognition Theory 7 lo; idiotypic network inspired experiments are subtle examples of ways in which S-F complexes can be made by interactions in vivo between Id (S) and anti-ld (F). For example, anti-ld reagents are usually allogeneic or xenogeneic F-components; 'syngeneic' anti-lds are sufficiently manipulated during their preparation (e.g., proteolysis in ascitic fluids) or purification (e.g., deamination during acid elution from affinity columns), that they are no longer intact (unaltered) self-components, i.e., they behave as S-F complexes in vivo. Similarly, the preparation of Ids themselves also creates S-F structures. Experiments involving the specificity of anti-ld reagents raise an intriguing question as to why the majority of Ids are defined by antibodies which recognize the D-region -of immunoglobulin H-chains. We note that, because of the extreme amino acid sequence diversity in the D-region, these antigenic determinants are present at such low concentration that they cannot competitively interfere
with a response to S induced by the cross-reactive S F immunogen. In contrast, V-, J-, and C-region determinants are present at relatively high concentration and this makes it difficult, though not impossible, to break tolerance. In conclusion, we recall that Jerne4 has exhorted us to look inside our own minds and there discover the true meaning of the immune system; we take his message to infer that when we stop and think, the 'perhaps never completely revealable' mystery of an idiotype network will be revealed, and, parenthetically, when we fail to think, the belief in an absurd immune system will prevail.
References 1 Jerne, N.K. (1974)Ann. Immunol. (Inst. Pasteur) 125C, 373-389 2 Jerne, N.K. (1976) The Harvey Lectures Series70, 93 110 3 Jerne,N.K. (1985)EMBOJ. 4, 847 852 4 Jerne, N.K. (1984)ImmunoL Rev. 79, 5-24 5 Coutinho, A. (1980)Ann. lmmunoL (Inst. Pasteur) 131D, 235-253 6 Forni, L. and Coutinho, A. (1981 ) in The Immune System Vol. 1 (Steinberg, C. and Lefkovitz, I. eds) pp. 21-27, S. Karger, Basel 7 Bretscher,P. and Cohn, M. (1970) Science 169, 1042-1049 8 Cohn, M. (1981) Cell• Immunol. 61,425-436; Cohn, M. (1981 ) in Immunoglobulin idiotypes and their expression, ICNUCLAsymposia on Molecular and Cellular Biology Vol. XX (Janeway, C., Sercarz, E.E.,Wigzell, H. and Fox, C.F. eds) pp. 881-849, Academic Press,New York 9 Cohn, M. (1982) Bulletin de I'lnstitut Pasteur 80, 343-380 10 Cohn, M. (1985) Biochimie 67, 9-27 Supported by Grant Number AI-05875 from the National Institutes of Health.
Does cyclosporine act in vivo as it does in vitro? Cyclosporine aborts the activation of lymphocytes in vitro before DNA synthesis begins, yet there is also compelling evidence that lymphocytes can become primed, Le. presumably profiferate in vivo, under the cover of immunosuppressive levels of the drug. Here, Gerry Klaus and Patricia Chisholm discuss the possibility that cyclosporine has a different mode of action in vivo and in vitro. The fungal metabolite cyclosporine (CS) has found widespread clinical acceptance as an immunosuppressant in organ transplantation. CS has significantly greater target specificity than traditional drugs such as azathioprine, or corticosteroids, since its action is largely restricted to cells of the lymphoid series (reviewed in Refs 1-3). This property is crucial to an understanding of the mode of action of CS and thus to the development of future generations of immunosuppressive drugs. The specificity of CS may also point to some unique features of growth control in lymphocytes. Consequently, many laboratories
Division of Immunology, National Institute for Medical Research, London NW7 1AA, UK; and Immunology Department, ChelseaCollege, Universityof London, London5W3 6LX, UK.
Gerry G.B. Klausand Patricia M. Chisholm have investigated the effect of CS on lymphocyte stimulation by antigens or mitogens in conjunction with growth factors, usually in vitro. The resultant fairly coherent• (if still incomplete) working model proposes that CS causes immunosuppression by aborting the activation of resting lymphocytes at an early stage, thus inhibiting the production of the T-cell growth factor, interleukin-2 (IL-2) by helper T (Th) cells 1-3. While evidence for this concept is compelling, various phenomena in vivo are not readily explained by this model and we discuss them here. In vitro inhibition of lymphocyte activation
CS exerts non-cytotoxic, reversible inhibitory effects on antigen or mitogen activation of quiescent lymphocytes. Its effects on the induction of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures have been analysed in some detail: The sequence of events believed to be involved in the generation of CTL may be summarized as follows. (1) Antigen is presented by an appropriate (~) 1986, Elsevier Science Publishers B.V., Amsterdam
0 1 6 7 - 4919/86/$02.00
101
-rostru MHC-compatible accessory cell [which also produces interleukin 1 (IL-1)], and this stimulates the expression of receptors for IL-2 on CTL precursors. (2) Th cells activated by antigen plus IL-1 secrete IL-2, which induces proliferation and differentiation of the precursors to functional CTL. CS seems to block the production of the two necessary cytokines IL-1 and IL-2 (Ref. 4) (and additional T-cellderived lymphokines), but leaves both T-cell proliferation in the presence of IL-2 and CTL effector function unaffected ~. In accord with this premise CS selectively inhibits the accumulation of mRNA coding for various lymphokines, including IL-2, in T cells without affecting mRNA synthesis in general s'6. Since mRNA for IL-2 is induced within a few hours of T-cell stimulation, the drug-sensitive phase of T-cell activation appears to be quite short. The effects of CS on the expression of IL-2 receptors (also an early event in activation) are not so clear-cut, but in most studies the drug produced at best only partial inhibition ~. We are not aware of investigations on the effects of CS on the levels of expression of high or tow affinity receptors for IL-2. CS was first thought to act only on T lymphocytes but it is now known to inhibit in-vitro activation of human and murine B cells by anti-lg antibodies 7-9, which are believed to mimic the early effects of antigen on B cells. Again, CS seems to block some as yet unidentified early event resulting from ligation of the surface Ig receptors 8'9, but not the later phases of B-cell growth and maturation controlled by T-cell-derived lymphokines. It seems fairly safe to conclude that, biochemically, the mode of action of CS on the activation of T cells and B cells is similar if not identical. It has now been established that ligation of antigen receptors on T and B lymphocytes provokes the rapid breakdown of ~nositol phospholipids in the plasma membrane, with consequent elevation of intracellular Ca2+, and activation of protein kinase C ~°'1~. The latter responses are believed to provide 'second messengers' for the initiation of lymphocyte activation. Cyclosporine seems to selectively block the activation of normal lymphocytes by agents which mobilize Ca 2+, namely ligands which cross-link antigen receptors, or Ca 2+ ionophores 12. Responses to polyclonal activators which do not provoke Ca2+ flux (phorbol esters, lipopolysac79 except charide, growth factors) are CS-resistant', perhaps in tumour cells6. However, CS does not abrogate either mitogen-induced phosphoinositide breakdown or Ca 2+ mobilization in T or B cells13'14. This indicates that the biochemical lesion produced by the drug in the Ca2÷-dependent signalling pathway must occur after second messenger generation and, in T cells, before the transcription of mRNA for various lymphokines. This conclusion accords with the demonstration that CS binds to the Ca2+-binding protein, calmodulin 15. However, this observation does not readily explain the target specificity of CS, since calmodulin is ubiquitous in eukaryotic cells and the lymphocyte protein has no known unique features.
102
Does CS inhibit lymphocyte proliferation in vivo? CS aborts lymphocyte activation at an early stage in vitro well before the onset of DNA synthesis. It is therefore widely, and reasonably, assumed that this also occurs in vivo, but this assumption has not been extensively tested. When it has been, the results have strongly
Immunology Today, vol. 7, No. 4, 1986
suggested that, in animals, T cells (and perhaps B cells) can become primed, and even proliferate in the presence of fully immunosuppressive levels of CS. This possibility was first suggested by the work of Bore116 who found that while CS suppressed the development of experimental allergic encephalomyelitis (EAE) in monkeys after primary immunization with myelin, the animals rapidly succumbed to the severe form of the disease (seen in primed individuals) when challenged with myelin after CS was stopped. It could be argued that this was due to rapid T-cell priming by a depot of antigen, and the phenomenon was not observed in EAE in the rat 17, where CS treatment of immunized donors abrogated the adoptive transfer of the disease to nai've recipients. In rabbits and mice primary antibody responses to T-dependent antigens are suppressed by CS, while secondary responses are not ~8'19. This is due to the rapid emergence of drug-resistant memory Th cells in primed mice 2° and rats21. Adoptive transfer experiments in mice demonstrated that CS blocked only the function of primary Th cells, and not T-cell priming: animals treated with CS during the priming period yielded normal (and under some treatment schedules even supranormal) numbers'of Th cells2°. Curiously, however, CS treatment did prevent the 'maturation' of the Th cells to the drug-resistant phenotype typical of fully-fledged memory cells. A similar phenomenon has been described in studies of the effects of CS on T cells mediating delayedtype hypersensitivity (DTH) reactions in mice 22'23. The clonal expansion of TDTH cells is unaffected by CS (Ref. 22) but, unlike T h cells, primed TDTH cells are functionally inhibited by C522,23, presumably because CS prevents the release of pro-inflammatory mediators. Similarly, CS suppresses the primary antibody responses of mice to type tl T-independent antigens such as DNP-ficol118, but it does not prevent the priming and clonal expansion of DNP-specific B cells by these antigens 24. There is evidence, therefore, that memory cells (both T and B) can be generated in mice which have immunosuppressive levels of CS. Since the establishment of memory presumably involves ctonal expansion, lymphocytes seem able to proliferate in these animals. A recent study 2s with rats has reinforced this conclusion. When parental strain lymphocytes are injected into F1 hybrid rats, donor cells reactive with host alloantigens are sequestered in the lymphoid tissues and do not appear in the thoracic duct lymph for at least 36 h. This negative selection was not impaired by treating the recipients with CS. However, the drug completely abolished the subsequent release of alloreactive blast cells into the thoracic duct. The spleens of these animals contained large numbers of lymphocytes incorporating 3H-thymidine in the T-dependent areas, but did not display the histological changes characteristic of a full-blown graft-versushost reaction (GVHR)2s. Further experiments have shown that intact F1 hybrid rats given parental lymphocytes and CS show an accelerated, severe GVHR when CS treatment is stopped (P. Chisholm, unpublished), suggesting again that sensitization had occurred under the cover of the drug. The interpretation of these experiments is that CS suppressed the GVHR and the release of alloreactive T cells into the circulation but did not affect the capacity of these cells to synthesize DNA. In short, the effects of CS in these experiments are exerted after the cells have entered S phase, perhaps even after the cells have divided.
Immunology Today, voL 7, No. 4, 1986
Conclusion
We are faced with the following paradox. If the growth of T cells is regulated by IL-2, their clonal expansion in vivo, must also be IL-2 dependent. CS effectively inhibits the production of IL-2 in vitro, yet in experimental animals, as we have discussed, T cells can apparently proliferate in vivo in the presence of concentrations of CS which abrogate T-cell effector functions. At present we have no satisfactory explanation for this paradox. Perhaps the effects of CS in vitro are due to the native peptide, while metabolites in vivo (presumably generated in the liver) have subtly different properties. There is no evidence for this attractive idea, except for a report that plasma from patients taking CS contains an agent distinct from the native molecule, which is immunosuppressive in vitro 26. The metabolites of CS have been extensively characterized, and none of the few that have been biologically tested has had the immunosuppressive activity in vitro of the native peptide 27. Priming under cover of CS has not been described in man. However, most patients taking CS are given additional immunosuppressive agents which may well complicate the picture. Clearly, much about CS remains unknown and some of its effects in vivo such as immune enhancement 18'28 and exacerbation of experimental autoimmunity 1 have also not been adequately explained. Such observations have been attributed to selective effects of the drug on ill-defined suppressor cells, although the capacity of CS to inhibit the activation of these cells in vitro is still unresolved 29'3°. We conclude that CS must produce more subtle perturbations of the immune network in vivo than suspected. Since available tissue culture systems cannot reproduce the microenvironments of lymphoid tissues further studies in vivo on the properties of CS are needed. References
1 Shevach,E.M. (1985)Ann. Rev. Immunol. 3,397~423 2Kahan, B.D. (1985) Transplant. Proc 17, 5-18 3 Klaus, G.G.B. (1981)lmmunoL Today2, 83-87 4 Bunjes, D., Hardt, C., Rollinghof, M. etaL (1981 ) Eur. J. Immunol. 11,657-66I 5 Kronke, M., Leonard, W.J., Depper, J.M. etaL (1984)Proc. NatlAcad. Sci. USA 81, 5214-5218
rostrun 6 Elliott, J.F., Lin, Y., Bleackley, R.C. etaL (1984) Science 226, 1439-1441 7 Dongworth, D.W. and Klaus, G.G.B. (1982)Eur. J. ImmunoL 12, 1018-1022 8 Muraguchi, A., Butler, J.L., Kehrl, J.H. etaL (1983)J. Exp. Med. 158, 690-702 9 Klaus, G.G.B. and Hawrylowicz, C.M. (1984) Eur. J. Irnmunol. 14, 250-254 10 Imboden, J.B. and Stobo, J.D. (1985)./. Exp. Med. 161, 446-456 11 Bijsterbosch, M.K., Meade, C.M, Turner, G.A. etaL (I985) Ce1141,999-1006 12 Kay, J.E., Benzie, C.R. and Borghetti, A.F. (1983) Immunology 50, 441-446 13 Metcalfe, S. (1984) Transplantation 38, 161-164 14 Bijsterbosch, M.K. and Klaus, G.G.B. (1985)Immunology 56, 435-440 15 Colombani, P.M., Robb, A. and Hess,A.D. (1985) Science 228, 337 339 16 Borel, J.F. (1981 ) in Transplantation and Clinical Immunology, Vol. Xlll (Touraine, J.L., Traeger, J. and Betuel, H. eds), p. 3, Excerpta Medica, Amsterdam 17 Bolton, C., AIIsop, G. and Cuzner, M.L. (1982) Clin. Exp. ImmunoL 47, 127-132 18 Kunkl, A. and Klaus, G.G.B. (1980)J. ImmunoL 125, 2526-2531 19 Lindsay,N., Harris, K.R., Norman, H.B. etaL (1980) Transplant. Proc 12, 252-254 20 Klaus, G.G.B. and Kunkl, A. (1983) Transplantation 36, 80-84 21 Homan, W.P., Fabre,J.W., Williams, K.A. etaL (1980) Transplantation 29,361-366 22 Milon, G., Truffa-Bachi, P., Shidani, B. etal. (1984)Ann. Immunol. (Inst. Pasteur) 135D, 237-245 23 Schiltknecht, E. and Ada, G.L. (1985) Cell. Immunol. 95, 340-348 24 Shidani, B, Colle, J.H., Motta, I. etaL (1983)Eur. J. ImmunoL 13, 359-363 25 Chisholm, P.M., Drayson, MT., Cox, J.H. etaL (1985) Eur. J. ImmunoL 15, 340-348 26 Fidelius, R.K. and Ferguson, R.M. (1983) Transplant. Proc. 15, 1921-1923 27 Maurer, G. (1985) Transplant. Proc 17, 19-26 28 Thomson, A.W., Moon, D.K., Inoue, Y. etal~ (1983) Immunology 48, 301-308 29 Hess,A.D. and Tutschka, P.J.(1980)J. ImmunoL 124, 2601-2607 30 Palacios, R. (1981) Cell. ImmunoL 61,453-462
103
A regulatory Id/anti-ld network in a real mouse suffering from the illusion of the 'completeness axiom' can only consist of a single pair of complementary and highly degenerate 'blobs'. Experimentalists who have been inspired by the idea of an idiotype network can claim to be dealing with real mice, and they may well point to repeated demonstrations that Ids and anti-lds can exert strong effects on the immune response. Aside from a few examples of experiments that have not been generalizable, there is no body of data that cannot be adequately (often better) explained by the Associative Recognition Theory 7. The effects of immunogenic complexes made up of self (S) and foreign (F) components (i.e., S-F) formed in vivo, have been thoroughly analysed within the framework of the Associative Recognition Theory 7 lo; idiotypic network inspired experiments are subtle examples of ways in which S-F complexes can be made by interactions in vivo between Id (S) and anti-ld (F). For example, anti-ld reagents are usually allogeneic or xenogeneic F-components; 'syngeneic' anti-lds are sufficiently manipulated during their preparation (e.g., proteolysis in ascitic fluids) or purification (e.g., deamination during acid elution from affinity columns), that they are no longer intact (unaltered) self-components, i.e., they behave as S-F complexes in vivo. Similarly, the preparation of Ids themselves also creates S-F structures. Experiments involving the specificity of anti-ld reagents raise an intriguing question as to why the majority of Ids are defined by antibodies which recognize the D-region -of immunoglobulin H-chains. We note that, because of the extreme amino acid sequence diversity in the D-region, these antigenic determinants are present at such low concentration that they cannot competitively interfere
with a response to S induced by the cross-reactive S F immunogen. In contrast, V-, J-, and C-region determinants are present at relatively high concentration and this makes it difficult, though not impossible, to break tolerance. In conclusion, we recall that Jerne4 has exhorted us to look inside our own minds and there discover the true meaning of the immune system; we take his message to infer that when we stop and think, the 'perhaps never completely revealable' mystery of an idiotype network will be revealed, and, parenthetically, when we fail to think, the belief in an absurd immune system will prevail.
References 1 Jerne, N.K. (1974)Ann. Immunol. (Inst. Pasteur) 125C, 373-389 2 Jerne, N.K. (1976) The Harvey Lectures Series70, 93 110 3 Jerne,N.K. (1985)EMBOJ. 4, 847 852 4 Jerne, N.K. (1984)ImmunoL Rev. 79, 5-24 5 Coutinho, A. (1980)Ann. lmmunoL (Inst. Pasteur) 131D, 235-253 6 Forni, L. and Coutinho, A. (1981 ) in The Immune System Vol. 1 (Steinberg, C. and Lefkovitz, I. eds) pp. 21-27, S. Karger, Basel 7 Bretscher,P. and Cohn, M. (1970) Science 169, 1042-1049 8 Cohn, M. (1981) Cell• Immunol. 61,425-436; Cohn, M. (1981 ) in Immunoglobulin idiotypes and their expression, ICNUCLAsymposia on Molecular and Cellular Biology Vol. XX (Janeway, C., Sercarz, E.E.,Wigzell, H. and Fox, C.F. eds) pp. 881-849, Academic Press,New York 9 Cohn, M. (1982) Bulletin de I'lnstitut Pasteur 80, 343-380 10 Cohn, M. (1985) Biochimie 67, 9-27 Supported by Grant Number AI-05875 from the National Institutes of Health.
Does cyclosporine act in vivo as it does in vitro? Cyclosporine aborts the activation of lymphocytes in vitro before DNA synthesis begins, yet there is also compelling evidence that lymphocytes can become primed, Le. presumably profiferate in vivo, under the cover of immunosuppressive levels of the drug. Here, Gerry Klaus and Patricia Chisholm discuss the possibility that cyclosporine has a different mode of action in vivo and in vitro. The fungal metabolite cyclosporine (CS) has found widespread clinical acceptance as an immunosuppressant in organ transplantation. CS has significantly greater target specificity than traditional drugs such as azathioprine, or corticosteroids, since its action is largely restricted to cells of the lymphoid series (reviewed in Refs 1-3). This property is crucial to an understanding of the mode of action of CS and thus to the development of future generations of immunosuppressive drugs. The specificity of CS may also point to some unique features of growth control in lymphocytes. Consequently, many laboratories
Division of Immunology, National Institute for Medical Research, London NW7 1AA, UK; and Immunology Department, ChelseaCollege, Universityof London, London5W3 6LX, UK.
Gerry G.B. Klausand Patricia M. Chisholm have investigated the effect of CS on lymphocyte stimulation by antigens or mitogens in conjunction with growth factors, usually in vitro. The resultant fairly coherent• (if still incomplete) working model proposes that CS causes immunosuppression by aborting the activation of resting lymphocytes at an early stage, thus inhibiting the production of the T-cell growth factor, interleukin-2 (IL-2) by helper T (Th) cells 1-3. While evidence for this concept is compelling, various phenomena in vivo are not readily explained by this model and we discuss them here. In vitro inhibition of lymphocyte activation
CS exerts non-cytotoxic, reversible inhibitory effects on antigen or mitogen activation of quiescent lymphocytes. Its effects on the induction of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures have been analysed in some detail: The sequence of events believed to be involved in the generation of CTL may be summarized as follows. (1) Antigen is presented by an appropriate (~) 1986, Elsevier Science Publishers B.V., Amsterdam
0 1 6 7 - 4919/86/$02.00
101
-rostru MHC-compatible accessory cell [which also produces interleukin 1 (IL-1)], and this stimulates the expression of receptors for IL-2 on CTL precursors. (2) Th cells activated by antigen plus IL-1 secrete IL-2, which induces proliferation and differentiation of the precursors to functional CTL. CS seems to block the production of the two necessary cytokines IL-1 and IL-2 (Ref. 4) (and additional T-cellderived lymphokines), but leaves both T-cell proliferation in the presence of IL-2 and CTL effector function unaffected ~. In accord with this premise CS selectively inhibits the accumulation of mRNA coding for various lymphokines, including IL-2, in T cells without affecting mRNA synthesis in general s'6. Since mRNA for IL-2 is induced within a few hours of T-cell stimulation, the drug-sensitive phase of T-cell activation appears to be quite short. The effects of CS on the expression of IL-2 receptors (also an early event in activation) are not so clear-cut, but in most studies the drug produced at best only partial inhibition ~. We are not aware of investigations on the effects of CS on the levels of expression of high or tow affinity receptors for IL-2. CS was first thought to act only on T lymphocytes but it is now known to inhibit in-vitro activation of human and murine B cells by anti-lg antibodies 7-9, which are believed to mimic the early effects of antigen on B cells. Again, CS seems to block some as yet unidentified early event resulting from ligation of the surface Ig receptors 8'9, but not the later phases of B-cell growth and maturation controlled by T-cell-derived lymphokines. It seems fairly safe to conclude that, biochemically, the mode of action of CS on the activation of T cells and B cells is similar if not identical. It has now been established that ligation of antigen receptors on T and B lymphocytes provokes the rapid breakdown of ~nositol phospholipids in the plasma membrane, with consequent elevation of intracellular Ca2+, and activation of protein kinase C ~°'1~. The latter responses are believed to provide 'second messengers' for the initiation of lymphocyte activation. Cyclosporine seems to selectively block the activation of normal lymphocytes by agents which mobilize Ca 2+, namely ligands which cross-link antigen receptors, or Ca 2+ ionophores 12. Responses to polyclonal activators which do not provoke Ca2+ flux (phorbol esters, lipopolysac79 except charide, growth factors) are CS-resistant', perhaps in tumour cells6. However, CS does not abrogate either mitogen-induced phosphoinositide breakdown or Ca 2+ mobilization in T or B cells13'14. This indicates that the biochemical lesion produced by the drug in the Ca2÷-dependent signalling pathway must occur after second messenger generation and, in T cells, before the transcription of mRNA for various lymphokines. This conclusion accords with the demonstration that CS binds to the Ca2+-binding protein, calmodulin 15. However, this observation does not readily explain the target specificity of CS, since calmodulin is ubiquitous in eukaryotic cells and the lymphocyte protein has no known unique features.
102
Does CS inhibit lymphocyte proliferation in vivo? CS aborts lymphocyte activation at an early stage in vitro well before the onset of DNA synthesis. It is therefore widely, and reasonably, assumed that this also occurs in vivo, but this assumption has not been extensively tested. When it has been, the results have strongly
Immunology Today, vol. 7, No. 4, 1986
suggested that, in animals, T cells (and perhaps B cells) can become primed, and even proliferate in the presence of fully immunosuppressive levels of CS. This possibility was first suggested by the work of Bore116 who found that while CS suppressed the development of experimental allergic encephalomyelitis (EAE) in monkeys after primary immunization with myelin, the animals rapidly succumbed to the severe form of the disease (seen in primed individuals) when challenged with myelin after CS was stopped. It could be argued that this was due to rapid T-cell priming by a depot of antigen, and the phenomenon was not observed in EAE in the rat 17, where CS treatment of immunized donors abrogated the adoptive transfer of the disease to nai've recipients. In rabbits and mice primary antibody responses to T-dependent antigens are suppressed by CS, while secondary responses are not ~8'19. This is due to the rapid emergence of drug-resistant memory Th cells in primed mice 2° and rats21. Adoptive transfer experiments in mice demonstrated that CS blocked only the function of primary Th cells, and not T-cell priming: animals treated with CS during the priming period yielded normal (and under some treatment schedules even supranormal) numbers'of Th cells2°. Curiously, however, CS treatment did prevent the 'maturation' of the Th cells to the drug-resistant phenotype typical of fully-fledged memory cells. A similar phenomenon has been described in studies of the effects of CS on T cells mediating delayedtype hypersensitivity (DTH) reactions in mice 22'23. The clonal expansion of TDTH cells is unaffected by CS (Ref. 22) but, unlike T h cells, primed TDTH cells are functionally inhibited by C522,23, presumably because CS prevents the release of pro-inflammatory mediators. Similarly, CS suppresses the primary antibody responses of mice to type tl T-independent antigens such as DNP-ficol118, but it does not prevent the priming and clonal expansion of DNP-specific B cells by these antigens 24. There is evidence, therefore, that memory cells (both T and B) can be generated in mice which have immunosuppressive levels of CS. Since the establishment of memory presumably involves ctonal expansion, lymphocytes seem able to proliferate in these animals. A recent study 2s with rats has reinforced this conclusion. When parental strain lymphocytes are injected into F1 hybrid rats, donor cells reactive with host alloantigens are sequestered in the lymphoid tissues and do not appear in the thoracic duct lymph for at least 36 h. This negative selection was not impaired by treating the recipients with CS. However, the drug completely abolished the subsequent release of alloreactive blast cells into the thoracic duct. The spleens of these animals contained large numbers of lymphocytes incorporating 3H-thymidine in the T-dependent areas, but did not display the histological changes characteristic of a full-blown graft-versushost reaction (GVHR)2s. Further experiments have shown that intact F1 hybrid rats given parental lymphocytes and CS show an accelerated, severe GVHR when CS treatment is stopped (P. Chisholm, unpublished), suggesting again that sensitization had occurred under the cover of the drug. The interpretation of these experiments is that CS suppressed the GVHR and the release of alloreactive T cells into the circulation but did not affect the capacity of these cells to synthesize DNA. In short, the effects of CS in these experiments are exerted after the cells have entered S phase, perhaps even after the cells have divided.
Immunology Today, voL 7, No. 4, 1986
Conclusion
We are faced with the following paradox. If the growth of T cells is regulated by IL-2, their clonal expansion in vivo, must also be IL-2 dependent. CS effectively inhibits the production of IL-2 in vitro, yet in experimental animals, as we have discussed, T cells can apparently proliferate in vivo in the presence of concentrations of CS which abrogate T-cell effector functions. At present we have no satisfactory explanation for this paradox. Perhaps the effects of CS in vitro are due to the native peptide, while metabolites in vivo (presumably generated in the liver) have subtly different properties. There is no evidence for this attractive idea, except for a report that plasma from patients taking CS contains an agent distinct from the native molecule, which is immunosuppressive in vitro 26. The metabolites of CS have been extensively characterized, and none of the few that have been biologically tested has had the immunosuppressive activity in vitro of the native peptide 27. Priming under cover of CS has not been described in man. However, most patients taking CS are given additional immunosuppressive agents which may well complicate the picture. Clearly, much about CS remains unknown and some of its effects in vivo such as immune enhancement 18'28 and exacerbation of experimental autoimmunity 1 have also not been adequately explained. Such observations have been attributed to selective effects of the drug on ill-defined suppressor cells, although the capacity of CS to inhibit the activation of these cells in vitro is still unresolved 29'3°. We conclude that CS must produce more subtle perturbations of the immune network in vivo than suspected. Since available tissue culture systems cannot reproduce the microenvironments of lymphoid tissues further studies in vivo on the properties of CS are needed. References
1 Shevach,E.M. (1985)Ann. Rev. Immunol. 3,397~423 2Kahan, B.D. (1985) Transplant. Proc 17, 5-18 3 Klaus, G.G.B. (1981)lmmunoL Today2, 83-87 4 Bunjes, D., Hardt, C., Rollinghof, M. etaL (1981 ) Eur. J. Immunol. 11,657-66I 5 Kronke, M., Leonard, W.J., Depper, J.M. etaL (1984)Proc. NatlAcad. Sci. USA 81, 5214-5218
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