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The cyclosporin-A-sensitive event in lymphocyte activation
622
19th FORUM IN IMMUNOLOGY
THE CYCWSPORIN-A-SENSITIVE EVENT IN LYMPHOCYTE ACTIVATION J.E. Kay
Biochemistry Laboratory and Centre for Medical Research, University of Sussex, Brighton BNl 9QG (UK) The immunosuppressive drug cyclosporin A (CsA) inhibits immunological responses primarily by blocking an early event in the induction of lymphocyte proliferation. Our studies on the activation of pig lymphocytes by mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (ConA) have defined this CsA-sensitive early event as one that is completed during the first 6-8 h after mitogen addition [1, 2]. The response of human lymphocytes to mitogens shows a similar progressive loss of sensitivity to CsA, but over a somewhat longer period [3]. The evidence below indicates that CsA acts by suppressing the responsiveness of lymphocytes to the transient early elevation of the cytoplasmic Ca 2 + concentration, one of the two signals that play key roles in the initiation of lymphocyte proliferation.
CsA prevents changes that occur in response to the Ca 2 + signal. There is now accumulating evidence that the initiation of lymphocyte proliferation requires the generation of two distinct intracellular signals [4, 5]. One signal is a rapid but transient elevation of the cytoplasmic Ca 2 + concentration [6] and the other can be replaced by phorbol esters such as TPA, which exert their effects by activating protein kinase C [7]. The metabolic changes induced in lymphocytes by TPA alone are insensitive to CsA [8], but activation by the calcium ionophore A23187 is particular1y sensitive to this drug [9]. CsA does not prevent the actual elevation of the cytoplasmic Ca 2 + concentration by mitogens [10, 11] or earlier related events such as the enhanced rate of turnover of phosphatidylinositides [12], but the completion of the CsA-sensitive
step in activation is dependent on the availability of extracellular Ca 2 + [2]. When pig lymphocytes are incubated with appropriate combinations of the Ca 2 + ionophore A23187 and the phorbol ester TPA, strong evidence of activation is seen (fig. 1). The combination of optimal concentrations of TPA with sub-mitogenic concentrations of A23187 is as effective as the strongest mitogenic lectins, and cannot be further enhanced by the addition of PHA or ConA. Co-incubation with submitogenic concentrations of both A23187 and TPA is nearly equally effective. Figure 1 shows that whatever proportion of the response is dependent on the presence of the Ca 2 + ionophore is inhibited by CsA - the extent of the inhibition observed thus depending on the combination used. It is notable that the A23187 concentrations that synergize most effectively with TPA are sub-mitogenic. A23187 alone is mitogenic only at concentrations that grossly increase'the total intracellular Ca 2 + and may well increase the cytoplasmic Ca 2 + to the unphysiologically high levels sufficient to activate protein kinase C even in the absence of TPA or its presumptive natural analogue diacylglycerol [7]. Other agents that can generate an increase in lymphocyte cytoplasmic Ca 2 + comparable to that induced by PHA or ConA are non-mitogenic or comparatively weakly mitogenic (e.g. wheat germ agglutinin or anti-T3 monoclonal antibodies such as OKT3), but synergize strongly with TPA [3, 13]. The intracellular Ca 2 + signal is thus necessary but not sufficient to induce activation in the absence of a second signal, the activation of protein kinase C. The synergistic effects of wheat germ agglutinin and OKT3 with TPA, like
MODE OF ACTION OF CYCLOSPORIN A
CWO)
623
5
o,.. I
><
4
E
c:i
o
3
2 1
o
0·5
5
50
ng/ml TPA FIG. 1. - Effect of CsA on the stimulation ofpig lymphocytes by TPA alone or in combina-
tion with A23I87.
Lymphocyte cultures were incubated with the concentration of TPA indicated, either alone (e, 0) or together with 100 ng/ml A23187 (_, D); 1 tJ.g/ml CsA was also added to some cultures (0, D) and lymphocyte stimulation was assessed by determination of the rate of incorporation of 35S-methionine into protein during a 4-h pulse terminating at 24 h, as previously described [2, 3].
those of A23187, are abolished by CsA ([3] and unpublished observations). When cells are pre-incubated with mitogenic combinations of A23187 and TPA, they complete the CsA-sensitive step in activation within 2-4 h. They still require the continued presence of stimulant for several further hours to generate a maximal response, but this can be provided by TPA alone. ConA and PHA are also effective, but their action after such pre-incubation is no longer sensitive to CsA [2]. However, pre-incubation with either A23187 or TPA alone is insufficient for the cells
to complete the CsA-sensitive stage of activation; the simultaneous presence of both signals is required. We have previously noted that the sensitivity of lymphocyte activation to inhibition by CsA is dependent on the mitogen used, the response to ConA being more sensitive than that to PHA [1]. This can now be accounted for by the hypothesis that effective mitogens must be able both to activate protein kinase C and to generate a cytoplasmic Ca 2 + signal, but that, just as different combinations of A23187 and TPA can produce similar degrees of lymphocyte
624
19th FORUM IN IMMUNOLOGY
activation while showing very different degrees of sensitivity to CsA, so different lymphocyte mitogens may cause different degrees of activation of the two intracellular signals. On this hypothesis, it would be predicted that PHA would be the more effective activator of protein kinase C. However, even ConA may activate this enzyme more strongly than the physiological signal to T lymphocytes through the T-cell receptor complex. Direct evidence for this is as yet lacking, but as lectins bind· to many different lymphocyte membrane receptors, it would not be surprising if they generated multiple intracellular signals. This conclusion that mitogens with similar overall effects may have differential effects on cytoplasmic Ca 2 + and protein kinase C may well also apply to B lymphocytes. Thus, the observation that B lymphocyte stimulation by anti-IgM is much more sensitive to CsA than activation by lipopolysaccharide [14] may be due to the former acting principally via a Ca 2 + signal and the latter via activation of protein kinase C, rather than to their effects being directed to separate CsA-sensitive and CsA-resistant B lymphocyte subpopulations. Indeed, CsA may prove a useful tool for the investigation of the extent to which lymphocyte responses are
dependent on a cytoplasmic Ca 2 + signal. The role of interleukin-2 and other Iymphokines. The combination of TPA and A23187 brings about its effects through the induction of interleukin-2 (lL-2) synthesis and the expression of IL-2 receptors, but the essential requirement for IL-2 itself is not bypassed [5, 15]. It is now well established that CsA strongly inhibits the production of IL-2 and several other lymphokines [16-18]. In the case of IL-2 at least, this inhibition is at the level of the transcription of its mRNA [19]. Studies with cell lines derived from T lymphocytes indicate that both a Ca 2 + signal and protein kinase C activation are essential for IL-2 induction [11, 20]. The question thus arises as to whether this inhibition of the synthesis of IL-2 and other lymphokines is the primary action of CsA or whether this is a secondary consequence of an earlier primary effect. The period required for mitogen-activated lymphocytes to become insensitive to CsA is much shorter than that required for maximal IL-2 synthesis, but sufficient for the production of significant quantities of IL-2 mRNA.
I. - Effect of 1 fLg/ml CsA on the stimulation of pig lymphocytes by 10 ng/ml TPA or 50 fLg/ml ConA, alone or in combination with 300 units/ml recombinant IL-2.
TABLE
Mitogen
None » TPA » ConA »
IL-2
+ + +
Stimulation was assessed as in figure 1.
Control
1 fLg/ml CsA
cpm.±SD
cpm.±SD
1,548 ± 77 2,484± 73 5,439± 136 lO,607± 44 1O,287±723 1O,742± 868
1,537 ± 135 2,281 ± 146 4,667± 70 1O,712± 167 4,982± 61 6,650± 128
MODE OF A CTION OF CYCLOSPORIN A In our hands, the effects of CsA on lymphocyte activation by mitogenic lectins are only marginally relieved by addition of pre-formed IL-2 or other lymphokines, indicating that inhibition of lymphokine synthesis is not the only mechanism by which activation is prevented by CsA. However, IL-2 can complement with high (but not low) TPA concentrations to give CsAresistant stimulation (table I), indicating that, with maximal protein kinase C activation, the only crucial role of the Ca 2 + signal is the induction of IL-2 mRNA synthesis. Under more physiological conditions, where the protein kinase C activation is weaker, the Ca 2 + signal has additional effects, and these too are sensitive to the action of CsA.
625
The role of calmodulin.
Colombani, Robb and Hess [21] have presented evidence implicating calmodulin as the target for CsA action. The inactivation by CsA of such a key mediator of the Ca2 + signal could provide an attractive explanation for our results, but as yet we and others [22-24] have been unable to reproduce the experiments on which Colombani et al.'s conclusion is based. It is also rather difficult to reconcile the physiological selectivity of CsA with an action directed against a protein with such a widespread involvement in the functions of many types of cells.
References. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
KAY, J.E. & BENZIE, CR., Immunology, 1983,49, 153. KAy, J.E. & BENZIE, e.R., Cell. Immunol., 1984,87,217. KAy, J.E. & BENZIE, CR., Immunology, 1986,57, 195. ISAKOv, N., SCHOLZ, W. & ALTMAN, A., Immunol. Today, 1986, 7, 271. TRUNEH, A., ALBERT, F., GOLSTEIN, P. & SCHMfIT-VERHULST, A.-M., Nature (Lond.), 1985, 313, 318. TSIEN, R.Y., POZZAN, T. & RINK, T.J., Nature (Lond.), 1982,295,68. NISHlZUKA, Y., Nature (Lond.), 1984, 308, 693. KAy, J.E., MEEHAN, R.T. & BENZIE, e.R., Immunol. Letters, 1983,7, 151. KAy, J.E., BENZIE, CR. & BORGHETII, A.F., Immunology, 1983,50,441. METCALFE, S., Transplant., 1984,38, 161. WISKOCIL, R., WEISS, A., IMBODEN, J., KAMIN-LEWIS, R. & STOBO, J., J. Immunol., 1985, 134, 3649. BUSTERBOSCH, M.K. & KLAUS, G.G.B., Immunology, 1985,56,435. GRIER, e.E. & MASTRO, A.M., J. Cell. Physiol., 1985, 124, 131. DONGWORTH, D.W. & KLAUS, G.G.B., Europ. J. Immunol., 1982, 12, 1018. ISAKOv, N. & ALTMAN, A., J. Immunol., 1985, 135,3674. BUNJES, D., HARDT, C, ROLLINGHOFF, M. & WAGNER, H., Europ. J. Immunol., 1981,11, 657. THOMSON, A.W., MOON, D.K., GECZY, e.L. & NELSON, D.S., Immunology, 1983,48,291. PALACIOS, R., MARTINEZ-MAZA, O. & DE LEY, M., Europ. J. Immunol., 1983, 13,221. KRONKE, M., LEONARD, W.J., DEPPER, J.M., ARYA, S.K., WONG-STAAL, F., GALLO, R.e., WALDMANN, T.A. & GREENE, w.e., Proc. nat. Acad. Sci. (Wash.), 1984,81,5214. ALBERT, F., HUA, C., TRUNEH, A., PIERRES, M. & SCHMITT-VERHULST, A.-M., J. Immunol., 1985, 134, 3649. eOLOMBANI, P.M., ROBB, A. & HESS, A.D., Science, 1985,228,337. HAlt, W.N., HARDING, M.W. & HANDSCHUMACHER, R.E., Science, 1986,233,987. LEGRUE, S.J., TURNER, R., WEISBRODT, N. & DEDMAN, J.R., Science, 1986,234,68. GUDGEON, M.e., BENZIE, CR. & KAY, J.E., Biochem. Soc. Trans., 1986, 14, 1050.
I thank Robin Benzie and Marianne Gudgeon for their collaboration and the Science & Engineering Research Council and the Medical Research Council for financial support.
19th FORUM IN IMMUNOLOGY
THE CYCWSPORIN-A-SENSITIVE EVENT IN LYMPHOCYTE ACTIVATION J.E. Kay
Biochemistry Laboratory and Centre for Medical Research, University of Sussex, Brighton BNl 9QG (UK) The immunosuppressive drug cyclosporin A (CsA) inhibits immunological responses primarily by blocking an early event in the induction of lymphocyte proliferation. Our studies on the activation of pig lymphocytes by mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (ConA) have defined this CsA-sensitive early event as one that is completed during the first 6-8 h after mitogen addition [1, 2]. The response of human lymphocytes to mitogens shows a similar progressive loss of sensitivity to CsA, but over a somewhat longer period [3]. The evidence below indicates that CsA acts by suppressing the responsiveness of lymphocytes to the transient early elevation of the cytoplasmic Ca 2 + concentration, one of the two signals that play key roles in the initiation of lymphocyte proliferation.
CsA prevents changes that occur in response to the Ca 2 + signal. There is now accumulating evidence that the initiation of lymphocyte proliferation requires the generation of two distinct intracellular signals [4, 5]. One signal is a rapid but transient elevation of the cytoplasmic Ca 2 + concentration [6] and the other can be replaced by phorbol esters such as TPA, which exert their effects by activating protein kinase C [7]. The metabolic changes induced in lymphocytes by TPA alone are insensitive to CsA [8], but activation by the calcium ionophore A23187 is particular1y sensitive to this drug [9]. CsA does not prevent the actual elevation of the cytoplasmic Ca 2 + concentration by mitogens [10, 11] or earlier related events such as the enhanced rate of turnover of phosphatidylinositides [12], but the completion of the CsA-sensitive
step in activation is dependent on the availability of extracellular Ca 2 + [2]. When pig lymphocytes are incubated with appropriate combinations of the Ca 2 + ionophore A23187 and the phorbol ester TPA, strong evidence of activation is seen (fig. 1). The combination of optimal concentrations of TPA with sub-mitogenic concentrations of A23187 is as effective as the strongest mitogenic lectins, and cannot be further enhanced by the addition of PHA or ConA. Co-incubation with submitogenic concentrations of both A23187 and TPA is nearly equally effective. Figure 1 shows that whatever proportion of the response is dependent on the presence of the Ca 2 + ionophore is inhibited by CsA - the extent of the inhibition observed thus depending on the combination used. It is notable that the A23187 concentrations that synergize most effectively with TPA are sub-mitogenic. A23187 alone is mitogenic only at concentrations that grossly increase'the total intracellular Ca 2 + and may well increase the cytoplasmic Ca 2 + to the unphysiologically high levels sufficient to activate protein kinase C even in the absence of TPA or its presumptive natural analogue diacylglycerol [7]. Other agents that can generate an increase in lymphocyte cytoplasmic Ca 2 + comparable to that induced by PHA or ConA are non-mitogenic or comparatively weakly mitogenic (e.g. wheat germ agglutinin or anti-T3 monoclonal antibodies such as OKT3), but synergize strongly with TPA [3, 13]. The intracellular Ca 2 + signal is thus necessary but not sufficient to induce activation in the absence of a second signal, the activation of protein kinase C. The synergistic effects of wheat germ agglutinin and OKT3 with TPA, like
MODE OF ACTION OF CYCLOSPORIN A
CWO)
623
5
o,.. I
><
4
E
c:i
o
3
2 1
o
0·5
5
50
ng/ml TPA FIG. 1. - Effect of CsA on the stimulation ofpig lymphocytes by TPA alone or in combina-
tion with A23I87.
Lymphocyte cultures were incubated with the concentration of TPA indicated, either alone (e, 0) or together with 100 ng/ml A23187 (_, D); 1 tJ.g/ml CsA was also added to some cultures (0, D) and lymphocyte stimulation was assessed by determination of the rate of incorporation of 35S-methionine into protein during a 4-h pulse terminating at 24 h, as previously described [2, 3].
those of A23187, are abolished by CsA ([3] and unpublished observations). When cells are pre-incubated with mitogenic combinations of A23187 and TPA, they complete the CsA-sensitive step in activation within 2-4 h. They still require the continued presence of stimulant for several further hours to generate a maximal response, but this can be provided by TPA alone. ConA and PHA are also effective, but their action after such pre-incubation is no longer sensitive to CsA [2]. However, pre-incubation with either A23187 or TPA alone is insufficient for the cells
to complete the CsA-sensitive stage of activation; the simultaneous presence of both signals is required. We have previously noted that the sensitivity of lymphocyte activation to inhibition by CsA is dependent on the mitogen used, the response to ConA being more sensitive than that to PHA [1]. This can now be accounted for by the hypothesis that effective mitogens must be able both to activate protein kinase C and to generate a cytoplasmic Ca 2 + signal, but that, just as different combinations of A23187 and TPA can produce similar degrees of lymphocyte
624
19th FORUM IN IMMUNOLOGY
activation while showing very different degrees of sensitivity to CsA, so different lymphocyte mitogens may cause different degrees of activation of the two intracellular signals. On this hypothesis, it would be predicted that PHA would be the more effective activator of protein kinase C. However, even ConA may activate this enzyme more strongly than the physiological signal to T lymphocytes through the T-cell receptor complex. Direct evidence for this is as yet lacking, but as lectins bind· to many different lymphocyte membrane receptors, it would not be surprising if they generated multiple intracellular signals. This conclusion that mitogens with similar overall effects may have differential effects on cytoplasmic Ca 2 + and protein kinase C may well also apply to B lymphocytes. Thus, the observation that B lymphocyte stimulation by anti-IgM is much more sensitive to CsA than activation by lipopolysaccharide [14] may be due to the former acting principally via a Ca 2 + signal and the latter via activation of protein kinase C, rather than to their effects being directed to separate CsA-sensitive and CsA-resistant B lymphocyte subpopulations. Indeed, CsA may prove a useful tool for the investigation of the extent to which lymphocyte responses are
dependent on a cytoplasmic Ca 2 + signal. The role of interleukin-2 and other Iymphokines. The combination of TPA and A23187 brings about its effects through the induction of interleukin-2 (lL-2) synthesis and the expression of IL-2 receptors, but the essential requirement for IL-2 itself is not bypassed [5, 15]. It is now well established that CsA strongly inhibits the production of IL-2 and several other lymphokines [16-18]. In the case of IL-2 at least, this inhibition is at the level of the transcription of its mRNA [19]. Studies with cell lines derived from T lymphocytes indicate that both a Ca 2 + signal and protein kinase C activation are essential for IL-2 induction [11, 20]. The question thus arises as to whether this inhibition of the synthesis of IL-2 and other lymphokines is the primary action of CsA or whether this is a secondary consequence of an earlier primary effect. The period required for mitogen-activated lymphocytes to become insensitive to CsA is much shorter than that required for maximal IL-2 synthesis, but sufficient for the production of significant quantities of IL-2 mRNA.
I. - Effect of 1 fLg/ml CsA on the stimulation of pig lymphocytes by 10 ng/ml TPA or 50 fLg/ml ConA, alone or in combination with 300 units/ml recombinant IL-2.
TABLE
Mitogen
None » TPA » ConA »
IL-2
+ + +
Stimulation was assessed as in figure 1.
Control
1 fLg/ml CsA
cpm.±SD
cpm.±SD
1,548 ± 77 2,484± 73 5,439± 136 lO,607± 44 1O,287±723 1O,742± 868
1,537 ± 135 2,281 ± 146 4,667± 70 1O,712± 167 4,982± 61 6,650± 128
MODE OF A CTION OF CYCLOSPORIN A In our hands, the effects of CsA on lymphocyte activation by mitogenic lectins are only marginally relieved by addition of pre-formed IL-2 or other lymphokines, indicating that inhibition of lymphokine synthesis is not the only mechanism by which activation is prevented by CsA. However, IL-2 can complement with high (but not low) TPA concentrations to give CsAresistant stimulation (table I), indicating that, with maximal protein kinase C activation, the only crucial role of the Ca 2 + signal is the induction of IL-2 mRNA synthesis. Under more physiological conditions, where the protein kinase C activation is weaker, the Ca 2 + signal has additional effects, and these too are sensitive to the action of CsA.
625
The role of calmodulin.
Colombani, Robb and Hess [21] have presented evidence implicating calmodulin as the target for CsA action. The inactivation by CsA of such a key mediator of the Ca2 + signal could provide an attractive explanation for our results, but as yet we and others [22-24] have been unable to reproduce the experiments on which Colombani et al.'s conclusion is based. It is also rather difficult to reconcile the physiological selectivity of CsA with an action directed against a protein with such a widespread involvement in the functions of many types of cells.
References. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
KAY, J.E. & BENZIE, CR., Immunology, 1983,49, 153. KAy, J.E. & BENZIE, e.R., Cell. Immunol., 1984,87,217. KAy, J.E. & BENZIE, CR., Immunology, 1986,57, 195. ISAKOv, N., SCHOLZ, W. & ALTMAN, A., Immunol. Today, 1986, 7, 271. TRUNEH, A., ALBERT, F., GOLSTEIN, P. & SCHMfIT-VERHULST, A.-M., Nature (Lond.), 1985, 313, 318. TSIEN, R.Y., POZZAN, T. & RINK, T.J., Nature (Lond.), 1982,295,68. NISHlZUKA, Y., Nature (Lond.), 1984, 308, 693. KAy, J.E., MEEHAN, R.T. & BENZIE, e.R., Immunol. Letters, 1983,7, 151. KAy, J.E., BENZIE, CR. & BORGHETII, A.F., Immunology, 1983,50,441. METCALFE, S., Transplant., 1984,38, 161. WISKOCIL, R., WEISS, A., IMBODEN, J., KAMIN-LEWIS, R. & STOBO, J., J. Immunol., 1985, 134, 3649. BUSTERBOSCH, M.K. & KLAUS, G.G.B., Immunology, 1985,56,435. GRIER, e.E. & MASTRO, A.M., J. Cell. Physiol., 1985, 124, 131. DONGWORTH, D.W. & KLAUS, G.G.B., Europ. J. Immunol., 1982, 12, 1018. ISAKOv, N. & ALTMAN, A., J. Immunol., 1985, 135,3674. BUNJES, D., HARDT, C, ROLLINGHOFF, M. & WAGNER, H., Europ. J. Immunol., 1981,11, 657. THOMSON, A.W., MOON, D.K., GECZY, e.L. & NELSON, D.S., Immunology, 1983,48,291. PALACIOS, R., MARTINEZ-MAZA, O. & DE LEY, M., Europ. J. Immunol., 1983, 13,221. KRONKE, M., LEONARD, W.J., DEPPER, J.M., ARYA, S.K., WONG-STAAL, F., GALLO, R.e., WALDMANN, T.A. & GREENE, w.e., Proc. nat. Acad. Sci. (Wash.), 1984,81,5214. ALBERT, F., HUA, C., TRUNEH, A., PIERRES, M. & SCHMITT-VERHULST, A.-M., J. Immunol., 1985, 134, 3649. eOLOMBANI, P.M., ROBB, A. & HESS, A.D., Science, 1985,228,337. HAlt, W.N., HARDING, M.W. & HANDSCHUMACHER, R.E., Science, 1986,233,987. LEGRUE, S.J., TURNER, R., WEISBRODT, N. & DEDMAN, J.R., Science, 1986,234,68. GUDGEON, M.e., BENZIE, CR. & KAY, J.E., Biochem. Soc. Trans., 1986, 14, 1050.
I thank Robin Benzie and Marianne Gudgeon for their collaboration and the Science & Engineering Research Council and the Medical Research Council for financial support.