The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin.

FK-506 inhibits Ca(2+)-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 related 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 (PPIase) 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 sufficient 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 L-685,818, 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 L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 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 PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.

Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.

The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) were previously shown to inhibit T cell stimulation through different mechanisms. FK-506 acts similarly to cyclosporin A (CsA) and prevents IL-2 production and IL-2R expression. RAP has little or no effect on these events but markedly impedes the response to IL-2. The present study was initiated to examine the possibility of a complementation between the immunosuppressive actions of RAP and FK-506 or CsA on various murine T cell responses. RAP potentiated the effect of CsA on proliferation and IL-2R expression in T cells stimulated with ionomycin + PMA. However, in the same system, RAP acted as a potent antagonist of FK-506 suppression. RAP also blocked FK-506- but not CsA-mediated inhibition of IL-2 mRNA induction. By using model systems sensitive to inhibition by RAP but not FK-506 we further demonstrated that FK-506 reciprocally behaves as an antagonist of RAP. In one such model, the stimulation of splenic T cells with IL-2 + PMA, FK-506, but not CsA, reversed the suppressive effect of RAP on proliferation. FK-506 also antagonized RAP-mediated inhibition with respect to the induction of Ly-6E Ag expression by IFN in YAC cells. To explore further the competition between the two macrolides at the cellular level, we performed binding experiments with a radiolabeled derivative of FK-506. Both FK-506 and RAP, but not CsA, inhibited the binding of this probe in YAC cells. Taken together, these data demonstrate that FK-506 and RAP antagonize each other's biologic activity and physically interact with a common receptor site(s) in T cells. Moreover, CsA acts at a site distinct from the cellular target(s) of FK-506 or RAP.

Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin.

FK-506 and the structurally related macrolide rapamycin (RAP) were investigated in comparison with cyclosporin A (CsA) for their immunosuppressive effects on murine T cells. All three agents suppressed the proliferation of splenic T cells triggered by lectins or antibodies to CD3 and Ly-6C. FK-506 or CsA also inhibited proliferation, IL-2 production, and IL-2R expression in splenic T cells activated with ionomycin + PMA. However, RAP minimally affected IL-2 production and IL-2R expression in these cells, although it reduced proliferation. Similarly, FK-506 and CsA, but not RAP, suppressed IL-2 production by activated DO.11.10 T hybridoma cells. In such a system, as well as in normal T cells stimulated with high ionomycin concentrations, FK-506 and CsA enhanced proliferation, indicating that they both abrogate negative signals associated with T cell activation. On the contrary, RAP diminished the autonomous proliferation of hybridoma cells, whereas FK-506 and CsA had little effect. The proliferative response induced in D10.G4 cells by IL-1 + ionomycin but not that induced by IL-1 + PMA was sensitive to inhibition by FK-506 and CsA. In contrast, RAP inhibited equally well both types of stimulation. Finally, T cell proliferation driven by IL-2 or IL-4 was found to be relatively resistant to FK-506 or CsA but sensitive to RAP. Altogether, these data demonstrate that FK-506 and CsA alter similar calcium-associated events of T cell activation and block T cell proliferation primarily by suppressing lymphokine production. RAP interferes with a different set of events and inhibits T cells by impairing their response to growth-promoting lymphokines.

A study of the correlation between cyclophilin binding and in vitro immunosuppressive activity of cyclosporine A and analogues.

In order to establish whether CyP is the pharmacologically relevant CsA receptor, the CyP binding v immunosuppressive activity was measured for an extensive, structurally varied group of CsA analogues. Overall, CyP binding was found to parallel immunosuppressive activity. Other than MeAla6-CsA, the few exceptions to the correlation could be ascribed to cellular metabolism. These results strongly implicate CyP or a related protein in the mechanism of action of cyclosporine.

Long-term passive enhancement of allogeneic skin grafts with monoclonal antibodies.

Monoclonal antibodies (mAb) to graft-specific class I or class II major histocompatibility antigens were tested for their ability to enhance the survival of allogeneic skin transplants. Mutant mouse strains were grafted with wild type tissue to restrict the antigenic differences being recognized. For allogeneic recognition of the class I antigen Ld, mutant BALB/c-H-2dm2 (dm2) mice were grafted with wild type BALB/cKh skin, and two dm2 anti-BALB/cKh mAb, 23-10-1 and 30-5-7, were tested for their ability to enhance. The anti-Ld antibody 23-10-1 (IgM) was found not to enhance the survival of BALB/c skin on dm2 mice. 30-5-7, however, an IgG2a antibody of indistinguishable specificity from 23-10-1, prolonged graft survival for approximately 5 days. For recognition of selected Iab determinants, mutant B6.C-H-2bm12 (bm12) mice were grafted with wild type B6/Kh skin, and mAb specific for the serological change(s) in bm12 were tested for their ability to enhance. The anti-Iab antibody 25-9-17 (IgG2a) was found not to enhance B6 grafts on bm12 mice. However, the enhancement seen with 25-9-17 using (C3H X bm12)F1 recipients was extraordinary, such that treated mice had a mean survival time three times that of the controls. Since 25-9-17 is of C3H origin, these results suggest that allotype (or possibly idiotype) compatibility is important in antibody enhancement. Another anti-Iab antibody 28-16-8 (IgM), also of C3H origin, failed to enhance a B6 graft on (C3H X bm 12)F1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)