CYCLOSPORIN A and the newly discovered immunosuppressant, FK-506, are potent inhibitors of T cell activation. In addition to their clinical importance in the prevention of allograft rejection, cyclosporin A and FK-506 represent important reagents for the study of the molecular mechanisms of lymphocyte activation. Cyclosporin A, a cyclic undecapeptide and FK-506, a macrolide, although chemically distinct, inhibit similar lymphocyte activation responses. The earliest responses inhibited in the T cell seem to be the expression of early phase T cell-activation genes for interleukins 2, 3 and 4, granulocyte-macrophage colony stimulating factor and gamma interferon. Although FK-506 and cyclosporin A seem to inhibit similar signal transduction processes, they do so be interacting with distinct cytosolic proteins. We report here the purification to homogeneity of a specific FK-506 binding protein that is distinct from the cyclosporin A-binding protein, cyclophilin. In addition, we show that this FK-506 binding protein, like cyclophilin, has peptidyl-prolyl isomerase activity.

SummaryThe role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca 2 +-activated potassium channels (Pc,c,) and voltage-gated potassium channels (PK,v) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (Ib2 and interferon 3') are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,C~ and Pg.v. we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for Pg, v. Theses studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca2+-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

SummaryFK-506 inhibits Ca2+-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with rehted biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis.trans peptidyl-prolyl isomerase activity (PPlase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not suf~cient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used b685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although b685,818 bound with high af~nity to FKBP-12 and inhibited its PPlase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, b685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPlase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.

No abstract

FK506, a neutral macrolide with immunosuppressive properties, was shown to selectively and rapidly inhibit the accumulation of IL-2 mRNA, as well as the mRNAs of other early (E) phase T cell activation genes such as IL-3, IL-4, GM-CSF, TNF alpha, IFN-gamma, and c-myc in activated human peripheral blood T cells. The activity of FK506, when compared to Cyclosporin A, another immunosuppressant, was 10 to 100x more potent in its ability to inhibit IL-2 mRNA synthesis. FK506 inhibited IL-2 mRNA accumulation in Con A, Con A plus PMA, Ionomycin plus PMA, anti-CD3, and anti-CD3 plus PMA activated T cells. Transcripts from other T cell gene classes such as the immediate early (IE) phase gene, c-fos, the late phase (L) genes, transferrin receptor, IL-2R alpha-chain, and TNF-beta, and the constitutive class genes glyceraldehyde-3-phosphate dehydrogenase and class I MHC HLA-B7 were not affected by FK506. The macrolide Rapamycin, which is structurally related to FK506, had no inhibitory effect on IE, E, L, or constitutive class mRNAs, but it appeared to increase the levels of the E-phase transcripts that were inhibited in FK506 treated T cells. The effect of FK506 on inducible genes in non-T and non-lymphoid human cells was studied in LPS-induced monocytes and PMA or IL-1 activated synovial fibroblasts. FK506 did not affect expression of the mRNAs for IL-1 alpha or IL-1 beta in human monocytes, or of stromelysin, collagenase, or TIMP in synovial fibroblasts. Nuclear run-off transcription studies indicate that FK506 inhibits transcription of the IL-2 gene. These studies suggest that Cyclosporin A and FK506 may effect a common early event in the T cell activation pathway.