Mixed agonist/antagonist activity of an FK-506-related immunosuppressant: biological and biochemical characterization.

FK-506 blocks T cell activation by preventing lymphokine gene transcription through formation of a complex with FKBP12 that inhibits calcineurin phosphatase activity. Immunosuppressive FK-506 analogs (agonists) have been generated whose potency correlates with calcineurin inhibition. Nonimmunosuppressive antagonist analogs have also been identified, including L-685,818, which binds to FKBP12 but does not inhibit calcineurin. We describe a novel property of FK-506 analog, characterized as a mixed agonist/antagonist immunosuppressive activity. It is displayed by L-688,617, the 32 O-methoxyethoxymethyl derivative of the agonist L-683,590 (C21-ethyl). Although it binds to FKBP12 similarly to L-683,590, L-688,617 incompletely suppressed T cell proliferation induced by optimal activation and enhanced that induced by supraoptimal activation. In the latter situation, L-688,617 suppressed IL-2 production only partially but blocked activation-driven cell death. Moreover, a 1000-fold molar excess of L-688,617 antagonized the immunosuppressive activity of L-683,590. L-688,617 inhibited calcineurin phosphatase activity in cells only partially. The unique agonist/antagonist activity of L-688,617 may therefore reflect its high affinity for FKBP12, combined with a reduced ability of the drug-FKBP12 complex to inhibit calcineurin function. However, in a cell-free system, L-688,617 completely blocked this function when a large excess of FKBP12 over calcineurin was present, suggesting that the intracellular concentration of FKBP12 may be a limiting factor that prevents full agonist activity of L-688,617 in cells.

Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma.

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.

Transforming growth factor beta 1 inhibits interleukin-1-induced but enhances ionomycin-induced interferon-gamma production in a T cell lymphoma: comparison with the effects of rapamycin.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.