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Mechanism of Action

Most immunosuppressants used in organ transplantation act primarily against the activation programs present within helper T cells (Fig. 2). Ciclosporin and tacrolimus act during the early, calcium-dependent, phase of T cell activation. At therapeutic concentrations, neither drug is cytotoxic and, in vitro, their effect can be reversed by the addition of IL-2). In vitro, to be effective in inhibiting T cell activation, ciclosporin or tacrolimus must be present at the time of T cell receptor ligation or soon afterwards; if the drug is administered more than 6 h after the activation stimulus there is little effect on T cell proliferation (Kay et al. 1983; Kumagai et al. 1988; Tocci et al. 1989).

Activation is initiated following the cognate interaction of an alloreactive T cell receptor with a MHC-peptide complex on an APC, coupled with a second signal involving the binding between costimulatory molecules such as B7-1 and 2 on the APC and CD28 on the T cell (Crabtree 1989; Sayegh and Turka 1998). This activates tyrosine kinases, which phosphorylate tyrosine residues of CD3 within the receptor complex (Fig. 4) (Shaw and Dustin 1997). Subsequent intracellular pathways are activated including the inositol triphosphate (IP3) and diacylglycerol (DAG) pathway. IP3 stimulates the release of calcium from the endoplasmic reticulum and opens a calcium channel in the membrane, producing an influx of calcium into the cell (Parekh et al. 1997; Shaw and Dustin 1997; Fierro et al. 2000).

Research using ciclosporin, tacrolimus and their analogues clarified the sequence of events that transduce this calcium signal into gene transcription within the T cell (Schreiber and Crabtree 1992; Liu 1993). Affinity matrices of FKBP and cyclophilin were used to identify ligands for complexes of FKBP-tacrolimus and cyclophilin-ciclosporin. Both were found to bind the same set of proteins in a calcium-dependent fashion (Liu et al. 1991). The target proteins were identified as the phosphatase calcineurin and the regulatory protein calmodulin (Klee et al. 1988; Liu 1993). Binding of each drug-immunophilin complex to calcineurin-calmodulin inhibited the phospha-tase activity of calcineurin (Liu 1993). In vitro, calcineurin activity was completely abolished by doses of ciclosporin or tacrolimus (hereafter calcineurin inhibitors or CNIs) that inhibited transcription of IL-2 (Fruman, Klee et al. 1992). Calcineurin consists of a catalytic domain coupled to a control region, a calmodulin-binding region and an autoinhibitory domain. Phosphatase activity is activated by the binding of calcium and calmodulin (Kincaid et al. 1986; Hubbard and Klee 1989).

Calcineurin dephosphorylates a number of substrates that play a role in intracellular regulation. The steps linking calcineurin phosphatase activity to the regulation of IL-2 are now understood, but the mechanisms that link this calcium-dependent pathway to effects that do not involve transcription and to the many adverse effects of CNIs are far less clear (Schreiber and Crabtree 1992). However, the correlation of immunosuppressive potency and nephrotoxicity within CNIs and their analogues supports the view that inhibition of calcineurin underlies the toxicity of these compounds (Sigal et al. 1991; Wiederrecht et al. 1993).

Calcineurin dephosphorylates the nuclear factor for activated T cells (NFAT) family of transcription factors. NFAT plays a key role in gene transcription within the activated lymphocyte (Shaw et al. 1988; Luo et al. 1996). NFAT1 is present in the cytoplasm of resting T cells and translocates into the nucleus in response to the calcium signal that follows T cell activation (Figs. 2 and 4) (Shaw et al. 1995; Loh et al. 1996). The event that initiates the nuclear import of NFAT1 is its dephosphorylation by calcineurin (Shaw et al. 1995; Luo et al. 1996). By inhibiting the translocation of NFAT to the nu-

Fig. 4 Mechanism of action of the calcineurin inhibitors. Binding of an MHC-peptide complex to its cognate T cell receptor (TCR) initiates a cascade of signalling events which constitute the early, calcium-dependent, phase of T cell activation. TCR-associated protein tyrosine kinases (PTKs) become activated and lead to the phosphorylation of phos-phatidylinositol phospholipase C (PPLC). PPLC then catalyses the breakdown of the membrane phospholipid phosphatidylinositol bisphosphate (PIP2) to form inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of calcium (Ca2+) from the endoplasmic reticulum (ER). The phosphatase calcineurin is then activated by the binding of calcium and the regulatory protein calmodulin. Calcineurin dephosphorylates regulatory proteins including the cytoplasmic form of the nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT can then be imported into the nucleus where it binds to the enhancer region of genes such as that for IL-2. DAG, in combination with calcium, activates protein kinase C (PKC), which phosphorylates other regulatory proteins. Other PTKs, activated by cognate binding of the TCR, phosphorylate substrates that activate guanosine triphosphate-binding proteins such as Ras. The Ras pathway links to a mitogen-activated protein (MAP) cascade (not shown) that ultimately leads to the transcription of Fos and the activation of Jun by phosphorylation. Fos and Jun-P combine to form the transcription factor activator protein (AP)-1 which binds to the enhancer region of the IL-2 gene. A number of factors can bind to the enhancer region of the IL-2 gene, including NFAT, AP-1, nuclear factor (NF)-kB and Oct-1; once bound, they serve to co-operatively activate the transcription of the gene. NFAT plays a key role in gene activation and the calcineurin inhibitors (CNIs) interfere with T cell activation by preventing its translocation into the cell nucleus. Ciclosporin and tacrolimus act as prodrugs which bind to their corresponding immunophilins (IMPN) to form a CNI-IMPN

cleus, calcineurin inhibition prevents the co-operation of NFAT with other transcription factors in enabling the expression of IL-2 and other cytokines (Durand et al. 1988; Wiederrecht et al. 1991; Schreiber and Crabtree 1992).

Experimentally, alternative pathways of T cell activation can bypass calcium-dependent activation of calcineurin, but their relevance to clinical allo-graft rejection and clinical immunosuppression remains uncertain (June et al. 1987; Lin et al. 1991). The clinical practice of combining a CNI with other types of immunosuppressant may mitigate the effect of such pathways (Banner and Lyster 2003).

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