REVIEW 45 Lin, R-H., Mamula, M.J., Hardin, J.A. and Janeway, C.A., Jr (1991) J. Exp. Med. 173, 1433-1439 46 Parish, C.R. (1972) Transplant. Rev. 13, 35-66 47 Walker, W.S. (1989) J. lmmunol. 143, 2142-2145 48 Blackman, M., Kappler, J. and Marrack, P. (1990) Science 248, 1335-1341 49 Burkly, L.C., Lo, D. and Flavell, R.A. (1990) Science 248, 1364-1368

50 Sarvetnick, N., Shiizuru, J., Liggitt, D. et al. (1990) Nature 346, 844-847 51 Oldstone, M.B., Nerenberg, M., Southern, P., Price, J. and Lweicki, H. (1991) Cell65, 319-331 52 Ohashi, P.S., Oehen, S., Buerki, K. et al. (1991) Cell 65, 305-317 53 Herman, A., Kappler, J.W., Marrack, P. and Pullen, A.M. (1991) Annu. Rev. Immunol. 9, 745-772

The mechanism of action of cyclosporin A and FK506 Stuart L. Schreiber and Gerald R. Crabtree CsA and FK506 are powerful suppressors of the immune system, most notably of T cells. They act at a point in activation that lies between receptor ligation and the transcription of early genes. Here, Stuart Schreiber and Gerald Crabtree review recent findings that indicate CsA and FK506 operate as prodrugs: they bind endogenous intracellular receptors, the immunophilins, and the resulting complex targets the protein phosphatase, calcineurin, to exert the immunosuppressive effect. The clinical utility of cyclosporin A (CsA) and FKS06 stems from their ability to prevent graft rejection following organ transplantation. These drugs exert their major therapeutic effects by inhibiting T-cell activation. The repression of an early step in T-cell activation apparently leads to a failure to activate the transcription of early genes, such as those encoding the cytokines, which normally function in coordinating the various cells involved in the immune response. Thus, from a primary effect largely but not wholly confined to T cells, a wide variety of secondary effects are mediated, leading to profound and therapeutically useful immunosuppression.

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While CsA and FK506 differ considerably in structure (Fig. 1), they have quite similar actions. They appear to act at a step distal to the cell membrane receptors and known second messengers, but proximal to late signaling events such as the transcriptional activation of early genes or the &granulation of mast cells. These findings led to the suggestion that the drugs can be used as effective probes for the actions of certain critical intracellular pathways of communication 1,2. Here, we review the data indicating that CsA and FK506 function by acting as prodrugs, only becoming active when complexed to endogenous, intracellular receptors or binding proteins named immunophilins, and that the target for the complex between drug and its cognate immunophilin is the protein phosphatase, calcineurin ~. Such is the case for CsA when bound to its receptor, cyclophilin 4,5, and for FK506, when bound to its receptor, FKBP 6-9. The immunosuppressive agent rapamycin (Fig. 1) appears to act in a similar manner, requiring binding to its cognate immunophilin, FKBP. However, present data indicate that the FKBP-rapamycin complex interacts with a molecule other than calcineurin J0.

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Biological actions of FK506 and CsA in T cells CsA was initially identified by screening for materials with antibiotic properties that were produced by fungi. It was later shown to be a powerful inhibitor of T-cell activation induced by mitogen or by mixed lymphocyte reaction 11,12. The observations by Borel and colleagues 11 that it was not toxic to cells at concentrations that completely blocked T-cell activation, and that it did not block the proliferation of other cell types, suggested that it might make a useful immunosuppressant. This prediction was, of course, accurate but, despite its widespread

© 1992, Elsm k'r Science Publishers Ltd, UK.

Immunology ro ay

136

Vol 13 No. 4 1992

REVIEW FK506/CsA sensitive

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Fig. 2. Proteins interacting with functional sequences within the IL-2 enhancer. The sequences identified as 'FKSOO/(~sA sensitive" are sensitive as judged by the ability of these drugs to block transcription directed by multimers of the indicated sites. The numbers u,der each site refer to the transcriptional activity of the II,-2 enhancer remaining after deleting the site"( clinical use, the mechanism of action of the drug remained elusive. Studies of its effects on T-cell activation indicate that CsA acts within several hours of the activation stimulus. If the drug is given within one hour of the activation stimuli there is a total block ofT-cell proliferation as well as virtually all of the late (more than three day) events associated with the function of T cells. In contrast, if the drug is given more than six hours after the activation stimulus there is little effect on T-cell proliferation induced in naive, resting T cells by either mitogen or antigen I~ i s. The period of susceptibility to these agents correlates well with the one to two hour commitment (or competence) period for T-cell activation, defined largely by studies of reversible antigen-receptor interactions I% The broad range of functions affected indicate that the drugs might be working very close to the critical molecules that establish the genetic program orchestrating later events in T-cell proliferation and activation. Mast cells and other cell types In mast cells, CsA and FK506 block degranulation as well as the transcriptional activation of several cytokine genes, such as interleukin 3 (IL-3) and IL-5 (Refs 17-20), and also the genes involved in leukotriene synthesis. Each of these events had the common requirement for an increase in the concentration of intracellular Ca 2+. Furthermore, the cytokine genes that are blocked in mast cells are largely the same as those blocked in T cells, suggesting an effect on a regulatory protein common to mast cells and T cells. CsA and FK506 have been reported to have effects on several other cell types including B cells, in which tumor necrosis factor c~ (TNF-c~) production is blocked by CsA at 100 blgml i (Ref. 21). In each case, an increase in the concentration of intracellular Ca 2+ is a requirement for those actions that are blocked by the immunosuppressants. Unfortunately, a tissue culture model and a precise molecular target for the toxic effects of CsA and FK506 on the kidney has not yet been developed and identified,

Immunology Today

respectively, but present evidence indicates that the target cell is associated with the vasculature. Over the past five to ten years a large body of essentially negative information has been collected, indicating that the drugs do not influence the actions of known molecules likely to have important roles in T-cell activation. The expression and activity of cell-surface molecules essential for T-Cell activation was unaffected by CsA and FK506. Furthermore, the drugs do not influence increases in intracellular Ca -,+ concentration, inositol phosphate metabolism, the activation of the tyrosine kinases or the tyrosine phosphatase, CD45, or the activities of a number of serine/threonine kinases-'-'. It was speculated that the drugs block a mvsterious and previously unknown step in T-cell activation, acting within the great black box between the cell membrane and the nucleus through which most developmental and cellular activation stimuli must pass, but which is almost totally unexplored. NF-AT and OAP: functional targets for FK506 and CsA Since CsA and FK506 were known to totally block the activation of many early genes in T cells z~ _,s a logical place to look for a molecular structure that received signals from the antigen receptor was among the proteins binding to the IL-2 enhancer (Fig. 2). These proteins appear to function cooperatively to activate transcription of the IL-2 gene. Several of them appear to play broad permissive functions in many tissues. For example, AP- 1, NF-KB and Oct- I are present in all tissues and respond to a wide variety of stimuli. In contrast, NF-AT plays a biologically restrictive function; its expression parallels that of the IL-2 gene>,~) and its binding site restricts expression of an indicator gene to activated T cells in intact transgenic animals ~l. By examining the ability of CsA and FK506 to inhibit the transcriptional competence of the individual factors, the NF-AT sites and the OAP (octamer-associated protein) site wcre simwn to be highly sensitive to the drug ~e ~'. Indeed, transcription controlled by these sites was reduced over 100-fold by

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REVIEW concentrations of the drugs that blocked T-cell activity but had no inhibitory effects on most other cell types. In contrast, the transcription factors AP-1 and NF-KB are either unaffected or only mildly affected in their ability to activate transcription, and this inhibitory effect on these factors occurs long after the 1L-2 gene is totally inhibited 32,-3s,36. These observations excluded AP-I or NF-KB from having a significant role in the actions of the drug. One report has indicated that AP-I is affected; however, these studies used an AP-1 site from the IL-2 gene that included the second NF-AT site (see above).

Intracellular binding molecules for CsA and FK506 Interest in the mechanism of action of these drugs was spurred by the observation that the major intracellular binding proteins for CsA, cyclophilin 4"s, was found to be identical to cis-trans peptidyl-prolyl isomerase (also known as a rotamase) and that the activity of the isomerase was blocked by the binding of CsA ~v,3~. This observation was soon followed by the discovery of a binding protein for FK506 and rapamycin, FK506binding protein (FKBP)