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Molecular mechanisms of the immunosuppressive action of cyclosporin A
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Pergamon
Int. J. lmmunopharmac., Vol. 19, No. 9/10, pp. 579 585, 1997 © 1998InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin Great Britain 0192~)561/98 $19.00+ .00
PII : S0192-0561(98)00004--6
M O L E C U L A R MECHANISMS OF THE IMMUNOSUPPRESSIVE ACTION OF CYCLOSPORIN A KLAUS RESCH* and MARTA SZAMEL Institute of Molecular Pharmacology, Medical School Hannover, Germany (Received for publication 16 January 1998) Keywords: T lymphocyte, T cell activation, signal transduction, cyclosporin A, immunosuppression
Since its introduction to clinical medicine in 1978, the fungal metabolite cyclosporin A (CyA) has developed into the standard drug for preventing rejection of organ transplants. It also has proven effective in the treatment of various autoimmune diseases, including rheumatoid arthritis. CyA causes serious side effects including nephro- or hepatotoxicity. Toxicity is more pronounced in situations where these organs are already affected by the underlying disease, this has limited its usefulness in systemic autoirnmune or chronic inflammatory diseases (Kahan, 1989; Altreya and Cassidy, 1991 ; Morris, 1991 ; Noble and Markham, 1995). Strategies to improve the benefit/risk ratio largely depend on the knowledge of the precise mechanism of action. The cellular targets of the immunosuppressive action of CyA have been well established (Ryffel, 1989; Morris, 1991; Baumann et al., 1992). The molecular mechanisms involved appeared to be largely resolved on the basis of experiments with cell lines (Chang et al., 1991 ; Sigal and Dumont, 1992; Bierer et al., 1993; Bram et al., 1993; Clipsone and Crabtree, 1992, 1993). However, it remained unclear whether they hold true for physiological human lymphocytes. As excellent reviews have been published recently, the current knowledge will only be briefly reviewed and emphasis will be laid on the findings with normal cells. In addition to CyA, more recently further nontoxic immunosuppressive drugs have been clinically approved, tacrolimus (FK 506) and sirolimus (rapamycin). Both are structurally macrolid antibiotics, while tacrolimus has a strikingly similar mechanism of action as compared to CyA (Schreiber and Crabtree, 1992) and also exerts overlapping toxicity such as nephrotoxicity, whereas that of sirolimus is different
(Chang et al., 1991). Both are only briefly discussed in the context with CyA.
1. THE CELLULAR TARGET OF CYA In contrast to the cytotoxic drugs such as cyclophosphamide, azathioprine, methotrexate, or antibodies to lymphocytic determinants, which all decrease the number of circulating lymphocytes, CyA and the macrolid immunosuppressants prevent their activation without affecting viability (Sharma et al., 1994). The activation of both B and T lymphocytes requires the participation of several cytokines, which serve as intercellular coactivators. CyA predominantly acts on T lymphocytes, preventing the synthesis of many (but not all) cytokines, most notably of the T cell growth factor interleukin-2 (IL2) (Rao, 1994; Jain et al., 1995 ; Serfling et al., 1995). Sirolimus, on the other hand interferes, with the IL-2 dependent cellular proliferation (Kuo et al., 1992). Inhibition of cytokine synthesis is not due to a general depression of protein synthesis, as other proteins involved in activation are not affected, an important example being the receptor for IL-2. CyA is a rather hydrophobic undekapeptide which can permeate easily through cellular membranes. It binds to intracellular proteins, which have been termed cyclophilins (Schreiber, 1991 ; Schreiber and Crabtree, 1992). Cyclophilins belong to a group of proteins--immunophilins--which are also targets of other immunsuppressants such as tacrolimus. Immunphilins are present in all eukaryotic cells in high concentrations. They are peptidyl-prolyl-cis-trans isomerases (PPIases), enzymes involved in the intracellular processing and especially folding of newly syn-
* Author to whom correspondence should be addressed. 579
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K. RESCH and M. SZAMEL
thesized proteins (Walsh et al., 1992). Although binding of CyA or tacrolimus to their respective immunophilins inhibits their enzymatic activity, it is clear that this is not responsible for the immunsuppressive mechanism, i.e. for inhibition of T cell activation (Bierer 1994: Fruman e t a / . , 1994: Schreibet, 1991). Inhibition of PPlase may be, however, associated with drug toxicity, as mutants of the lower eukaryotes Neurospora or Saccharomyces, lacking cyclophilin, proved to be resistant to the otherwise toxic action of CyA (Schreiber, 1991). Experiments with a great number of structural analogues of CyA (and FK506) revealed that there are distinct domains of the cyclic peptides, some responsible for the binding to immunophilins, the second mediating its immunosuppression (Schreiber and Crabtree, 1992: Rayat ez al., 1993: Bierer, 1994, 1995). This favoured a model that the complex of CyA/immunophilin (or tacrolimus/immunophilin) favours the interaction with a further cellular component, thereby inhibiting activation. It should be mentioned that CyA also binds to less characterised proteins, some of which seem to be located in the plasma membrane, although the biological relevance is unknown (Erlanger, 1992).
2. MOLECULAR MECHANISMS OF CYA ACTION 2.1. Regulation ¢~f l L - 2 gene expression The best understood mechanism involved in immunosuppression consists in the inhibition of the synthesis of cytokines, especially IL-2. Accordingly, regulation of the IL-2 gene expression has been studied most extensively and prototypically, although minor differences may occur with other cytokines. The complete gene structure for IL-2, including the structural and regulatory (promoter/enhancer) regions, are known in several animal species as well as in human beings (Crabtree and Clipstone, 1994: Jain e t a / . , 1995; Rao, 1994; Settling et al., 1995). Experiments with reporter gene constructs containing the regulatory sequences or parts of them revealed that multiple regulatory elements control the IL-2 gene. These include transcription factors which participate in the regulation of several genes in many cells such as NF,,B, and factors which appear to be more restricted to lymphocytes, like the nuclear factors of activated T-lymphocytes, NFAT (Bierer et al., 1993 : Baeuerle and Henkel, 1994; Crabtree and Clipstone, 1994; Jain et al., 1995). 2.2. The N F A T / a m i / v To date at least lk)ur different NFAT genes (NFATp = NFATI. NFAT,. NFAT3 or NFAT4/X)
have been cloned, and they constitute a related but divergent gene family. The hallmark of NFAT activity is its inducibility by agents that increase intracellular calcium flux (Crabtree and Clipstone, 1994; Serfling et ell., 1995). NFATp mRNA is expressed constitutively in many different lymphoid cell types and preexisting NFATp protein translocates to the nucleus upon stimulation of cells. NFAT~. mRNA is expressed less widely, and at least in some cell types (human NK cells, some Jurkat T cell clones) it is not expressed unless the cells are stimulated (with immune complexes or with phorbol ester and ionomycin). NFAT3 mRNA was found at significant levels in several organs, but little or none was seen in spleen, thymus or peripheral blood lymphocytes. NFAT4 mRNA is elevated in thymus and is detectable in various organs. In Jurkat T cells it is expressed constitutively (Crabtree, 1994). In freshly isolated human peripheral blood lymphocytes (T cells) NFATp ( = N F A T 1 ) protein is constitutively expressed, while synthesis of NFAT~ can be induced by ionomycin. Trace amounts of NFAT4/X were expressed in unstimulated cells, and their level did not increase upon activation. NFAT3 protein was not observed under any conditions. Exposure of cells to phorbol ester+anti-CD28 did not induce NFAT,. suggesting that at least under these conditions IL-2 synthesis was independent of NFAT~, (Lyakh et al., 1997). Thus in contrast to several T cell lines, NFATp appears to be the predominant transcription factor of this family in normal human T lymphocytes. Thus. NFAT family members are regulated in a different way in peripheral blood lymphocytes as compared to cell lines.
2.3. N F A T assembly a n d the inte~/i'rence hy C v A The characteristic feature of NFAT proteins is that they bind cooperatively with Fos and Jun family proteins to the distal IL-2 promoter NFAT site (Jain et al., 1993). Overexpression of most Fos and Jun family members augments NFAT-driven transcription. whereas a dominant negative Jun (but not Fos) inhibits this process (Rooney et al., 1995). Although there is little evidence so far for selective cooperation of individual NFAT proteins with specific Fos/Jun dimers, they may require Fos/Jun dimers for optimal activity. Overexpression ofc-Fos resulted in increased synthesis of IL-2 in stimulated murine T cells (Ochi el al., 1994). Post-translational modification of Fos and Jun proteins is also likely to influence their role in 1L2 gene expression. A serine/threonine-specific protein kinase phosphorylating c-Fos in t,itro is rapidly activated in T cells stimulated with anti-CD3 (Nel et al.,
lmmunosuppressive Action of Cyclosporin A 1994). Jun amino terminal kinases, JNKs (belonging to the MAP kinase family) activate c-Jun by phosphorylating its N-terminal activation domain. Interestingly, J N K activity is differently regulated in T cells as compared to other cells. In T-lymphocytes a combined stimulation with phorbol ester and ionomycin is required for maximal JNK activation (Su et al., 1994; Gupta et al., 1996). To interact with the Fos/Jun dimer and thereby forming the active transcription factor complex the cytoplasmic N F A T components--in human peripheral T-lymphocytes predominantly N F A T p - - h a v e to enter the nucleus. Dephosphorylation of N F A T p precedes translocation to the nucleus and is associated with an increase in its affinity to the DNA. N F A T p is, however, rapidly rephosphorylated and again localizes to the cytoplasm (Loh et al., 1996), requiring a sustained dephosphorylation in the cytosol to maintain optimal transcription factor activity. The enzyme responsible for dephosphorylation of N F A T p is the Ca 2+ dependent phosphoprotein phosphatase calcineurin. In T-lymphocytes, cystosolic Ca 2+ is raised rapidly, both by the release from internal stores in the endoplasmic reticulum through inositol trisphosphate and the subsequent opening of plasma membrane channels. Importantly, an elevated Ca 2+-level is maintained in the cells for at least several hours (Bram and Crabtree, 1994 ; Fraser et al., 1993 ; Timmermann et al., 1996). CyA or tacrolimus complexed to their respective immunophilins bind calcineurin and thereby inhibit its activity towards phosphorylated proteins (Crabtree and Clipstone, 1994). As a consequence the cytosolic N F A T components cannot be dephosphorylated and enter the nucleus. Thus no active transcription factor in combination with the Fos/Jun dimer can be formed. In accordance with this mechanism overexpression of calcineurin in T-lymphocytic cell lines such as Jurkat markedly decreases their sensitivity towards the inhibitory action of CyA (O'Keefe et al., 1992). As shown more recently overexpression and constitutive localisation of N F A T in the nucleus rendered the IL-2 enhancer resistant to CyA and FK506 (Timmermann e t al., 1996). Both types of experiments support the idea that inhibition of calcineurin and its effects on the nuclear translocation of N F A T are important mechanisms for the immunosuppressive action of these drugs. Collecting evidence suggests other targets of calcineurin that might be J N K family members and IkBa. Calcineurin also acts in synergy with phorbol esters to inactivate IkB (Suet al., 1994 ; Frantz et al., 1994). A recent, unexpected finding even questioned the activatory role of NFAT1 in vivo at least in mice. Cells
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from N F A T I - / - mice showed increased immune responses, suggesting that N F A T 1 might exert a negative regulatory influence in cells of the immune system (Xanthoudakis et al., 1996).
2.4. Activation o f protein kinase C: an additional taryet Jor Cy A It is well established that mitogenic activation of T-lymphocytes requires the participation of protein kinase C (PKC). This includes the transcription of the IL-2 gene as well as the synthesis and expression of its high affinity receptors (Weiss and Littman 1994; Szamel and Resch, 1995). One of the key experiments supporting this is the direct activation of PKC by phorbol esters, which together with a rise in cytosolic Ca 2+ is sufficient to mimic activation ofT lymphocytes as obtained by the ligation of the surface T cell receptor. In our own experiments with human peripheral blood T lymphocytes, both monoclonal antibodies directed to the TCR/CD3 complex or a combination ofphorbol ester and the calcium ionophore ionomycin led to a similar extent of cellular poliferation (Szamel et al., 1993). CyA, as documented by many groups, completely blocked the proliferative response of human T lymphocytes to an anti-CD3 monoclonal antibody in a dose dependent way with an ICs0 of about 8 ng/ml which was completely reversed by the addition of recombinant human IL-2. In contrast, lymphocyte proliferation induced by phorbol ester proved to be resistant to inhibition by CyA (Szamel et al., 1993). This suggested that PKC or mechanisms leading to its activation constitute a target for the action of CyA. As CyA did not interfere directly with PKC activity, this pointed to a signalling event between the T cell receptor and this enzyme as the molecular target. PKC comprises a family of at least 11 isoenzymes which can be grouped into different families, i.e. "classical" PKCs (~, 31, f12 and 7) being activated by calcium and diacylglycerol, calcium independent or novel PKCs, (like 6, E, r/, 0,/0 and atypical isoforms lacking the diacylglycerol-binding domain ( ~, 1/2) (Hug and Sarre, 1993). Of these--with the exception of the brain specific isoenzyme PKC-7--all have been shown to be present in T-lymphocytes. Activation of resting peripheral T lymphocytes by T-cell receptor antibodies directed against monomorphic determinants (such as BMA 31) induced a bimodal activation of PKC peaking initially at about 10 min, followed by a long-lasting activation starting at about 60 rain and reaching its maximum at 90 min of activation (Szamel et al., 1989, 1993, 1997). Several groups including our own have
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shown that for tile induction of IL-2 gene transcription a long-lasting activation of PKC is required, whereas short term activation, e.g. by a single administration of a short lived membrane permeable diglyceride is sufficient to elicit IL-2 receptor synthesis and expression (Szamel and Resch, 1995). Interestingly, at 10 rain of activation only PKC-~ and 0 were activated as measured by the concomitant translocation to the plasma membrane whereas at 90 min of activation only PKC-fl, -6 and -t: were activated and translocated. Other PKC-isoenzymes were not affected (Szamel et al., 1998). CyA added prior to the stimulating anti T-cell receptor antibody completely prevented activation and translocation of PKC-fl, whereas activation of PKC-c~ was left imact (Szamel et al., 1993). As CyA completely suppresses IL-2 synthesis from these findings it appears unlikely that PKC-c~ (and -0) are implicated in 1L-2 synthesis, which is also supported by their only transient activation. Recently we found that cholera toxin, which blocks the so called PI response, i.e. the breakdown of phosphatidylinositol bisphosphate into inositol trisphosphate and diacylglycerol, completely prevented activation of PKC-~ while the long-lasting activation of PKC-fl was not affected. Simultaneously the expression of highaffinity IL-2 receptors was completely blocked, whereas IL-2 synthesis proceeded normally. Thus these experiments established a link between PI response, activation of PKC-c~ (and -0), and expression of IL-2 receptors (Szamel et al., 1997). The selective inhibition of the activation of PKC-fl by CyA on the other hand suggested its role in the induction of IL-2 synthesis. To prove a link between PKC isoenzymes and IL-2 gene expression, neutralizing antibodies against various PKC-isoenzymes were introduced into human peripheral lymphocytes by means of electroporation. While antibodies against PKC-cc and -0 were without effect, antibodies against PKC-fl, -6 or -~, partially inhibited 1L-2 synthesis. Of these, antibodies against PKC-fl were most effective in inhibiting IL-2 secretion or IL-2 m R N A induction by about 60 to 70% (Szamel et al., 1993, 1997 ; Szamel et al., 1998). These data establish the link between Tcell receptor, PKC-fl (and/or -6, -t:), and IL-2 gene expression. They also corroborate the importance of the blocking of these PKC isoenzymes as a mechanism for the immunsuppressive action of CyA in normal cells. CyA does not interfere with the enzymatic activity itself of any of the PKC isoenzymes. Thus the molecular target of the immunosuppressive agent as pointed out above--must be a signal transduction process upstream of PKC activation. Of these, as evident from
the experiments with cholera toxin, the degradation of phosphatidylinositol bisphosphate is not involved. Diacylglycerols necessary for the activation of PKC can also be generated from other phospholipid species, most notably from phosphatidylcholine. Indeed, phosphatidylcholine-specific phospholipases are activated upon stimulation of human T-lymphocytes (Mollinedo et al., 1994). in resting T-lymphocytes phosphatidylcholine contains predominantly saturated or monounsaturated fatty acids, which lead to diacylglycerols with only weak PKC stimulating efficacy (Berry and Nishizuka, 1990; Szamel and Resch, 1995). It has been shown that diacylglycerols with strong PKC activating capacity must contain polyunsaturated fatty acids (Berry and Nishizuka, 1990). In stimulated lymphocytes the fatty acid moieties of membrane phospholipids are rapidly exchanged, leading to an increase of the content of polyunsaturated fatty acids. We have shown earlier that CyA completely blocked the activation of membrane phospholipid fatty acid metabolism (Szamel et al., 1993). Model ./or the inhibition 0/ the interleukin-2 gene expression by CyA. As shown on Fig. 1 binding of appropriate antigens MHC bound peptides at the surface of accessory cells--to T-cell receptors, or in model situations monoclonal anti T-cell receptor antibodies lead to activation of intracellular signal transduction cascades which finally result in the expression of several genes. Expression of IL-2 and its receptor play a crucial role in proliferation and concomitant clonal expansion of T-lymphocytes. The functional structure of the IL-2 gene has been largely elucidated. Its promotor/enhancer region contains sequences for several wide-spread transcription factors such as NF~B, as well as for more T-cell specific factors. The active transcription factor of the N F A T site contains two components, one of which is rapidly induced upon activation and represents AP-I, composed of members of the Fos and Jun family. The other is located in an inactive, phosphorylated form in the cytosol and activated by desphosphorylation, upon which it translocates to the nucleus ; in normal T lymphocytes it is NFATp. Initial receptor associated activation events consist in the recruitment of a protein tyrosine-kinase, ZAP 70, which phosphorylates and thereby activates phospholipase C (Crabtree and Clipstone, 1994; Weiss and Littman, 1994). This enzyme cleaves phosphatidylinositol bisphosphates to diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytosolic Ca 2+ which--amongst other processes activates the phosphoprotein phosphatase calcineurin, which desphosphorylates N F A T p and thus con-
Immunosuppressive Action of Cyclosporin A
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F
\ I
I
I
~z
I
Fig. 1. Inhibition by cyclosporin A of signal transduction steps leading to interleukin-2 expression. Abbreviations : TCR : Tcell antigen receptor; DAG : 1,2-diacylglycerol ; ERK : extracellular signal regulated kinase ; 1L-2 : interleukin-2 ; Ins P3 : inositol-trisphosphate ; MAP kinase : mitogen activated protein kinase ; MEK : MAPK/ERK kinase ; NFAT : nuclear factor (of) activated T cells ; PC : phosphatidylcholine ; PI : phosphatidylinositol ; PKC : protein kinase C ; PLC)' : phospholipase C 7.
tributes to the IL-2 gene expression. CyA and tacrolimus both after binding to their respective immunophilins complex with calcineurin block its activity. This constitutes one target of the immunosuppression by the drugs. U p o n activation the T-cell receptor initiates several protein kinase dependent signalling cascades. The best
studies consists in the activation of Raf-1 and the subsequent phosphorylation and activation of M E K ( = M A P kinase kinase) and the M A P kinases, E R K t and 2. The M A P kinases then are involved in the synthesis and/or activation of transcription factors. Raf- 1 can be activated by binding to p21 Ras (Cantrell, 1994, 1996). Alternative pathways consist in the acti-
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K. RESCH and M. SZAMEL
vation of protein kinase(s) C, which also can enter the M A P kinase pathway at more distant sites, e.g. M E K . O u r own experiments suggested that specific P K C isoenzymes, p r e d o m i n a n t l y PKC-/~, are involved in the induction of IL-2 gene expression. The coupling of this P K C isoenzyme to the T cell receptor has not been established. Available evidence points to phosphatidylcholine and phosphtyatidyl-choline-specific phospholipases being involved. CyA completely blocks the activation o f PKC-/~, the P K C isoform linked to IL-2 synthesis. Experiments, including o u r own, suggest that in physiological h u m a n blood lymphocytes CyA blocks the induction o f c - J u n , one com-
p o n e n t of AP-I in the nucleus (Granelli-Piperno, 1990). Both mechanisms, inhibition of the activation of the cytosolic c o m p o n e n t N F A T (a calcineurin d e p e n d e n t mechanism), as well as prevention of the induction of c-Jun, an essential part of the nuclear c o m p o n e n t AP1 (a P K C - d e p e n d e n t mechanism), explain why CyAand similarly t a c r o l i m u s - - a r e such powerful and rather T-lymphocyte selective immunosuppressive drugs. The competent typing assistance of Mrs K. Erdogan is gratefully acknowledged.
Aeknowledqements
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(1995) T-cell antigen receptor-induced signal-transduction pathways. Activation and function of protein kinases C in T lymphocytes. Eur. J. Biochem. 228, 1-15. Szamel, M., Ebel, U., Uciechowski, P., Kaever, V. and Resch, K. (1997) T cell receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-~. Biochim. Biophys. Acta. 1356, 237-248. Timmermann, L. A., Clipstone, A., Ho, S. N., Northrop, J. P. and Crabtree, G. R. (1996) Rapid shuttling of NT-AT in discrimination of Ca 2+ signals and immunosuppression. Nature 383, 837-840. Walsh, C. T., Zydowsky, L. D. and McKeon, F. D. (1992) Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade o f T cell signal transduction. J. Biol. Chem. 267, 13115-13118. Weiss, A. and Littman, D. R. (1994) Signal transduction by lymphocyte antigen receptors. Cell 76, 263-274. Xanthoudakis, S., Viola, J. P., Shaw, K. T., Luo, C., Wallace, J. D., Bozza, P. T., Curran, T. and Rao, A. (1996) An enhanced immune response in mice lacking the transcription factor NFATI. Science 272, 892 895.
Pergamon
Int. J. lmmunopharmac., Vol. 19, No. 9/10, pp. 579 585, 1997 © 1998InternationalSocietyfor Immunopharmacology Publishedby ElsevierScienceLtd. Printedin Great Britain 0192~)561/98 $19.00+ .00
PII : S0192-0561(98)00004--6
M O L E C U L A R MECHANISMS OF THE IMMUNOSUPPRESSIVE ACTION OF CYCLOSPORIN A KLAUS RESCH* and MARTA SZAMEL Institute of Molecular Pharmacology, Medical School Hannover, Germany (Received for publication 16 January 1998) Keywords: T lymphocyte, T cell activation, signal transduction, cyclosporin A, immunosuppression
Since its introduction to clinical medicine in 1978, the fungal metabolite cyclosporin A (CyA) has developed into the standard drug for preventing rejection of organ transplants. It also has proven effective in the treatment of various autoimmune diseases, including rheumatoid arthritis. CyA causes serious side effects including nephro- or hepatotoxicity. Toxicity is more pronounced in situations where these organs are already affected by the underlying disease, this has limited its usefulness in systemic autoirnmune or chronic inflammatory diseases (Kahan, 1989; Altreya and Cassidy, 1991 ; Morris, 1991 ; Noble and Markham, 1995). Strategies to improve the benefit/risk ratio largely depend on the knowledge of the precise mechanism of action. The cellular targets of the immunosuppressive action of CyA have been well established (Ryffel, 1989; Morris, 1991; Baumann et al., 1992). The molecular mechanisms involved appeared to be largely resolved on the basis of experiments with cell lines (Chang et al., 1991 ; Sigal and Dumont, 1992; Bierer et al., 1993; Bram et al., 1993; Clipsone and Crabtree, 1992, 1993). However, it remained unclear whether they hold true for physiological human lymphocytes. As excellent reviews have been published recently, the current knowledge will only be briefly reviewed and emphasis will be laid on the findings with normal cells. In addition to CyA, more recently further nontoxic immunosuppressive drugs have been clinically approved, tacrolimus (FK 506) and sirolimus (rapamycin). Both are structurally macrolid antibiotics, while tacrolimus has a strikingly similar mechanism of action as compared to CyA (Schreiber and Crabtree, 1992) and also exerts overlapping toxicity such as nephrotoxicity, whereas that of sirolimus is different
(Chang et al., 1991). Both are only briefly discussed in the context with CyA.
1. THE CELLULAR TARGET OF CYA In contrast to the cytotoxic drugs such as cyclophosphamide, azathioprine, methotrexate, or antibodies to lymphocytic determinants, which all decrease the number of circulating lymphocytes, CyA and the macrolid immunosuppressants prevent their activation without affecting viability (Sharma et al., 1994). The activation of both B and T lymphocytes requires the participation of several cytokines, which serve as intercellular coactivators. CyA predominantly acts on T lymphocytes, preventing the synthesis of many (but not all) cytokines, most notably of the T cell growth factor interleukin-2 (IL2) (Rao, 1994; Jain et al., 1995 ; Serfling et al., 1995). Sirolimus, on the other hand interferes, with the IL-2 dependent cellular proliferation (Kuo et al., 1992). Inhibition of cytokine synthesis is not due to a general depression of protein synthesis, as other proteins involved in activation are not affected, an important example being the receptor for IL-2. CyA is a rather hydrophobic undekapeptide which can permeate easily through cellular membranes. It binds to intracellular proteins, which have been termed cyclophilins (Schreiber, 1991 ; Schreiber and Crabtree, 1992). Cyclophilins belong to a group of proteins--immunophilins--which are also targets of other immunsuppressants such as tacrolimus. Immunphilins are present in all eukaryotic cells in high concentrations. They are peptidyl-prolyl-cis-trans isomerases (PPIases), enzymes involved in the intracellular processing and especially folding of newly syn-
* Author to whom correspondence should be addressed. 579
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K. RESCH and M. SZAMEL
thesized proteins (Walsh et al., 1992). Although binding of CyA or tacrolimus to their respective immunophilins inhibits their enzymatic activity, it is clear that this is not responsible for the immunsuppressive mechanism, i.e. for inhibition of T cell activation (Bierer 1994: Fruman e t a / . , 1994: Schreibet, 1991). Inhibition of PPlase may be, however, associated with drug toxicity, as mutants of the lower eukaryotes Neurospora or Saccharomyces, lacking cyclophilin, proved to be resistant to the otherwise toxic action of CyA (Schreiber, 1991). Experiments with a great number of structural analogues of CyA (and FK506) revealed that there are distinct domains of the cyclic peptides, some responsible for the binding to immunophilins, the second mediating its immunosuppression (Schreiber and Crabtree, 1992: Rayat ez al., 1993: Bierer, 1994, 1995). This favoured a model that the complex of CyA/immunophilin (or tacrolimus/immunophilin) favours the interaction with a further cellular component, thereby inhibiting activation. It should be mentioned that CyA also binds to less characterised proteins, some of which seem to be located in the plasma membrane, although the biological relevance is unknown (Erlanger, 1992).
2. MOLECULAR MECHANISMS OF CYA ACTION 2.1. Regulation ¢~f l L - 2 gene expression The best understood mechanism involved in immunosuppression consists in the inhibition of the synthesis of cytokines, especially IL-2. Accordingly, regulation of the IL-2 gene expression has been studied most extensively and prototypically, although minor differences may occur with other cytokines. The complete gene structure for IL-2, including the structural and regulatory (promoter/enhancer) regions, are known in several animal species as well as in human beings (Crabtree and Clipstone, 1994: Jain e t a / . , 1995; Rao, 1994; Settling et al., 1995). Experiments with reporter gene constructs containing the regulatory sequences or parts of them revealed that multiple regulatory elements control the IL-2 gene. These include transcription factors which participate in the regulation of several genes in many cells such as NF,,B, and factors which appear to be more restricted to lymphocytes, like the nuclear factors of activated T-lymphocytes, NFAT (Bierer et al., 1993 : Baeuerle and Henkel, 1994; Crabtree and Clipstone, 1994; Jain et al., 1995). 2.2. The N F A T / a m i / v To date at least lk)ur different NFAT genes (NFATp = NFATI. NFAT,. NFAT3 or NFAT4/X)
have been cloned, and they constitute a related but divergent gene family. The hallmark of NFAT activity is its inducibility by agents that increase intracellular calcium flux (Crabtree and Clipstone, 1994; Serfling et ell., 1995). NFATp mRNA is expressed constitutively in many different lymphoid cell types and preexisting NFATp protein translocates to the nucleus upon stimulation of cells. NFAT~. mRNA is expressed less widely, and at least in some cell types (human NK cells, some Jurkat T cell clones) it is not expressed unless the cells are stimulated (with immune complexes or with phorbol ester and ionomycin). NFAT3 mRNA was found at significant levels in several organs, but little or none was seen in spleen, thymus or peripheral blood lymphocytes. NFAT4 mRNA is elevated in thymus and is detectable in various organs. In Jurkat T cells it is expressed constitutively (Crabtree, 1994). In freshly isolated human peripheral blood lymphocytes (T cells) NFATp ( = N F A T 1 ) protein is constitutively expressed, while synthesis of NFAT~ can be induced by ionomycin. Trace amounts of NFAT4/X were expressed in unstimulated cells, and their level did not increase upon activation. NFAT3 protein was not observed under any conditions. Exposure of cells to phorbol ester+anti-CD28 did not induce NFAT,. suggesting that at least under these conditions IL-2 synthesis was independent of NFAT~, (Lyakh et al., 1997). Thus in contrast to several T cell lines, NFATp appears to be the predominant transcription factor of this family in normal human T lymphocytes. Thus. NFAT family members are regulated in a different way in peripheral blood lymphocytes as compared to cell lines.
2.3. N F A T assembly a n d the inte~/i'rence hy C v A The characteristic feature of NFAT proteins is that they bind cooperatively with Fos and Jun family proteins to the distal IL-2 promoter NFAT site (Jain et al., 1993). Overexpression of most Fos and Jun family members augments NFAT-driven transcription. whereas a dominant negative Jun (but not Fos) inhibits this process (Rooney et al., 1995). Although there is little evidence so far for selective cooperation of individual NFAT proteins with specific Fos/Jun dimers, they may require Fos/Jun dimers for optimal activity. Overexpression ofc-Fos resulted in increased synthesis of IL-2 in stimulated murine T cells (Ochi el al., 1994). Post-translational modification of Fos and Jun proteins is also likely to influence their role in 1L2 gene expression. A serine/threonine-specific protein kinase phosphorylating c-Fos in t,itro is rapidly activated in T cells stimulated with anti-CD3 (Nel et al.,
lmmunosuppressive Action of Cyclosporin A 1994). Jun amino terminal kinases, JNKs (belonging to the MAP kinase family) activate c-Jun by phosphorylating its N-terminal activation domain. Interestingly, J N K activity is differently regulated in T cells as compared to other cells. In T-lymphocytes a combined stimulation with phorbol ester and ionomycin is required for maximal JNK activation (Su et al., 1994; Gupta et al., 1996). To interact with the Fos/Jun dimer and thereby forming the active transcription factor complex the cytoplasmic N F A T components--in human peripheral T-lymphocytes predominantly N F A T p - - h a v e to enter the nucleus. Dephosphorylation of N F A T p precedes translocation to the nucleus and is associated with an increase in its affinity to the DNA. N F A T p is, however, rapidly rephosphorylated and again localizes to the cytoplasm (Loh et al., 1996), requiring a sustained dephosphorylation in the cytosol to maintain optimal transcription factor activity. The enzyme responsible for dephosphorylation of N F A T p is the Ca 2+ dependent phosphoprotein phosphatase calcineurin. In T-lymphocytes, cystosolic Ca 2+ is raised rapidly, both by the release from internal stores in the endoplasmic reticulum through inositol trisphosphate and the subsequent opening of plasma membrane channels. Importantly, an elevated Ca 2+-level is maintained in the cells for at least several hours (Bram and Crabtree, 1994 ; Fraser et al., 1993 ; Timmermann et al., 1996). CyA or tacrolimus complexed to their respective immunophilins bind calcineurin and thereby inhibit its activity towards phosphorylated proteins (Crabtree and Clipstone, 1994). As a consequence the cytosolic N F A T components cannot be dephosphorylated and enter the nucleus. Thus no active transcription factor in combination with the Fos/Jun dimer can be formed. In accordance with this mechanism overexpression of calcineurin in T-lymphocytic cell lines such as Jurkat markedly decreases their sensitivity towards the inhibitory action of CyA (O'Keefe et al., 1992). As shown more recently overexpression and constitutive localisation of N F A T in the nucleus rendered the IL-2 enhancer resistant to CyA and FK506 (Timmermann e t al., 1996). Both types of experiments support the idea that inhibition of calcineurin and its effects on the nuclear translocation of N F A T are important mechanisms for the immunosuppressive action of these drugs. Collecting evidence suggests other targets of calcineurin that might be J N K family members and IkBa. Calcineurin also acts in synergy with phorbol esters to inactivate IkB (Suet al., 1994 ; Frantz et al., 1994). A recent, unexpected finding even questioned the activatory role of NFAT1 in vivo at least in mice. Cells
581
from N F A T I - / - mice showed increased immune responses, suggesting that N F A T 1 might exert a negative regulatory influence in cells of the immune system (Xanthoudakis et al., 1996).
2.4. Activation o f protein kinase C: an additional taryet Jor Cy A It is well established that mitogenic activation of T-lymphocytes requires the participation of protein kinase C (PKC). This includes the transcription of the IL-2 gene as well as the synthesis and expression of its high affinity receptors (Weiss and Littman 1994; Szamel and Resch, 1995). One of the key experiments supporting this is the direct activation of PKC by phorbol esters, which together with a rise in cytosolic Ca 2+ is sufficient to mimic activation ofT lymphocytes as obtained by the ligation of the surface T cell receptor. In our own experiments with human peripheral blood T lymphocytes, both monoclonal antibodies directed to the TCR/CD3 complex or a combination ofphorbol ester and the calcium ionophore ionomycin led to a similar extent of cellular poliferation (Szamel et al., 1993). CyA, as documented by many groups, completely blocked the proliferative response of human T lymphocytes to an anti-CD3 monoclonal antibody in a dose dependent way with an ICs0 of about 8 ng/ml which was completely reversed by the addition of recombinant human IL-2. In contrast, lymphocyte proliferation induced by phorbol ester proved to be resistant to inhibition by CyA (Szamel et al., 1993). This suggested that PKC or mechanisms leading to its activation constitute a target for the action of CyA. As CyA did not interfere directly with PKC activity, this pointed to a signalling event between the T cell receptor and this enzyme as the molecular target. PKC comprises a family of at least 11 isoenzymes which can be grouped into different families, i.e. "classical" PKCs (~, 31, f12 and 7) being activated by calcium and diacylglycerol, calcium independent or novel PKCs, (like 6, E, r/, 0,/0 and atypical isoforms lacking the diacylglycerol-binding domain ( ~, 1/2) (Hug and Sarre, 1993). Of these--with the exception of the brain specific isoenzyme PKC-7--all have been shown to be present in T-lymphocytes. Activation of resting peripheral T lymphocytes by T-cell receptor antibodies directed against monomorphic determinants (such as BMA 31) induced a bimodal activation of PKC peaking initially at about 10 min, followed by a long-lasting activation starting at about 60 rain and reaching its maximum at 90 min of activation (Szamel et al., 1989, 1993, 1997). Several groups including our own have
582
K. RESCH and M. SZAMEL
shown that for tile induction of IL-2 gene transcription a long-lasting activation of PKC is required, whereas short term activation, e.g. by a single administration of a short lived membrane permeable diglyceride is sufficient to elicit IL-2 receptor synthesis and expression (Szamel and Resch, 1995). Interestingly, at 10 rain of activation only PKC-~ and 0 were activated as measured by the concomitant translocation to the plasma membrane whereas at 90 min of activation only PKC-fl, -6 and -t: were activated and translocated. Other PKC-isoenzymes were not affected (Szamel et al., 1998). CyA added prior to the stimulating anti T-cell receptor antibody completely prevented activation and translocation of PKC-fl, whereas activation of PKC-c~ was left imact (Szamel et al., 1993). As CyA completely suppresses IL-2 synthesis from these findings it appears unlikely that PKC-c~ (and -0) are implicated in 1L-2 synthesis, which is also supported by their only transient activation. Recently we found that cholera toxin, which blocks the so called PI response, i.e. the breakdown of phosphatidylinositol bisphosphate into inositol trisphosphate and diacylglycerol, completely prevented activation of PKC-~ while the long-lasting activation of PKC-fl was not affected. Simultaneously the expression of highaffinity IL-2 receptors was completely blocked, whereas IL-2 synthesis proceeded normally. Thus these experiments established a link between PI response, activation of PKC-c~ (and -0), and expression of IL-2 receptors (Szamel et al., 1997). The selective inhibition of the activation of PKC-fl by CyA on the other hand suggested its role in the induction of IL-2 synthesis. To prove a link between PKC isoenzymes and IL-2 gene expression, neutralizing antibodies against various PKC-isoenzymes were introduced into human peripheral lymphocytes by means of electroporation. While antibodies against PKC-cc and -0 were without effect, antibodies against PKC-fl, -6 or -~, partially inhibited 1L-2 synthesis. Of these, antibodies against PKC-fl were most effective in inhibiting IL-2 secretion or IL-2 m R N A induction by about 60 to 70% (Szamel et al., 1993, 1997 ; Szamel et al., 1998). These data establish the link between Tcell receptor, PKC-fl (and/or -6, -t:), and IL-2 gene expression. They also corroborate the importance of the blocking of these PKC isoenzymes as a mechanism for the immunsuppressive action of CyA in normal cells. CyA does not interfere with the enzymatic activity itself of any of the PKC isoenzymes. Thus the molecular target of the immunosuppressive agent as pointed out above--must be a signal transduction process upstream of PKC activation. Of these, as evident from
the experiments with cholera toxin, the degradation of phosphatidylinositol bisphosphate is not involved. Diacylglycerols necessary for the activation of PKC can also be generated from other phospholipid species, most notably from phosphatidylcholine. Indeed, phosphatidylcholine-specific phospholipases are activated upon stimulation of human T-lymphocytes (Mollinedo et al., 1994). in resting T-lymphocytes phosphatidylcholine contains predominantly saturated or monounsaturated fatty acids, which lead to diacylglycerols with only weak PKC stimulating efficacy (Berry and Nishizuka, 1990; Szamel and Resch, 1995). It has been shown that diacylglycerols with strong PKC activating capacity must contain polyunsaturated fatty acids (Berry and Nishizuka, 1990). In stimulated lymphocytes the fatty acid moieties of membrane phospholipids are rapidly exchanged, leading to an increase of the content of polyunsaturated fatty acids. We have shown earlier that CyA completely blocked the activation of membrane phospholipid fatty acid metabolism (Szamel et al., 1993). Model ./or the inhibition 0/ the interleukin-2 gene expression by CyA. As shown on Fig. 1 binding of appropriate antigens MHC bound peptides at the surface of accessory cells--to T-cell receptors, or in model situations monoclonal anti T-cell receptor antibodies lead to activation of intracellular signal transduction cascades which finally result in the expression of several genes. Expression of IL-2 and its receptor play a crucial role in proliferation and concomitant clonal expansion of T-lymphocytes. The functional structure of the IL-2 gene has been largely elucidated. Its promotor/enhancer region contains sequences for several wide-spread transcription factors such as NF~B, as well as for more T-cell specific factors. The active transcription factor of the N F A T site contains two components, one of which is rapidly induced upon activation and represents AP-I, composed of members of the Fos and Jun family. The other is located in an inactive, phosphorylated form in the cytosol and activated by desphosphorylation, upon which it translocates to the nucleus ; in normal T lymphocytes it is NFATp. Initial receptor associated activation events consist in the recruitment of a protein tyrosine-kinase, ZAP 70, which phosphorylates and thereby activates phospholipase C (Crabtree and Clipstone, 1994; Weiss and Littman, 1994). This enzyme cleaves phosphatidylinositol bisphosphates to diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytosolic Ca 2+ which--amongst other processes activates the phosphoprotein phosphatase calcineurin, which desphosphorylates N F A T p and thus con-
Immunosuppressive Action of Cyclosporin A
583
F
\ I
I
I
~z
I
Fig. 1. Inhibition by cyclosporin A of signal transduction steps leading to interleukin-2 expression. Abbreviations : TCR : Tcell antigen receptor; DAG : 1,2-diacylglycerol ; ERK : extracellular signal regulated kinase ; 1L-2 : interleukin-2 ; Ins P3 : inositol-trisphosphate ; MAP kinase : mitogen activated protein kinase ; MEK : MAPK/ERK kinase ; NFAT : nuclear factor (of) activated T cells ; PC : phosphatidylcholine ; PI : phosphatidylinositol ; PKC : protein kinase C ; PLC)' : phospholipase C 7.
tributes to the IL-2 gene expression. CyA and tacrolimus both after binding to their respective immunophilins complex with calcineurin block its activity. This constitutes one target of the immunosuppression by the drugs. U p o n activation the T-cell receptor initiates several protein kinase dependent signalling cascades. The best
studies consists in the activation of Raf-1 and the subsequent phosphorylation and activation of M E K ( = M A P kinase kinase) and the M A P kinases, E R K t and 2. The M A P kinases then are involved in the synthesis and/or activation of transcription factors. Raf- 1 can be activated by binding to p21 Ras (Cantrell, 1994, 1996). Alternative pathways consist in the acti-
584
K. RESCH and M. SZAMEL
vation of protein kinase(s) C, which also can enter the M A P kinase pathway at more distant sites, e.g. M E K . O u r own experiments suggested that specific P K C isoenzymes, p r e d o m i n a n t l y PKC-/~, are involved in the induction of IL-2 gene expression. The coupling of this P K C isoenzyme to the T cell receptor has not been established. Available evidence points to phosphatidylcholine and phosphtyatidyl-choline-specific phospholipases being involved. CyA completely blocks the activation o f PKC-/~, the P K C isoform linked to IL-2 synthesis. Experiments, including o u r own, suggest that in physiological h u m a n blood lymphocytes CyA blocks the induction o f c - J u n , one com-
p o n e n t of AP-I in the nucleus (Granelli-Piperno, 1990). Both mechanisms, inhibition of the activation of the cytosolic c o m p o n e n t N F A T (a calcineurin d e p e n d e n t mechanism), as well as prevention of the induction of c-Jun, an essential part of the nuclear c o m p o n e n t AP1 (a P K C - d e p e n d e n t mechanism), explain why CyAand similarly t a c r o l i m u s - - a r e such powerful and rather T-lymphocyte selective immunosuppressive drugs. The competent typing assistance of Mrs K. Erdogan is gratefully acknowledged.
Aeknowledqements
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