Probing T-cell signal transduction pathways with the immunosuppressive drugs,FK-506 and rapamycin

In addition to their clinical utility in tissue transplantation the immunosuppressive agents FK-506 (Prograf) and rapamycin, have proven to be valuable tools for gaining insight into the biochemistry of T-cell activation. The findings that the protein phosphatase calcineurin and cell cycle control are key elements in T-cell activation and proliferation are the direct result of investigations into the mechanism of action of FK-506 and rapamycin and provide potentially novel therapeutic targets.     

Inhibition of T-lymphocyte activation by the immunosuppressive drug FK-506.

Nanamolar concentrations of the immunosuppressive drug FK-506 inhibit the induction of T-lymphocyte proliferation by the lectins concanavalin A (Con A) and phytohaemagglutinin (PHA). Activation by Con A is more sensitive to inhibition than the response to PHA. FK-506 inhibits an early Ca2+-dependent step in the activation process, and its effects are not reversible by the addition of recombinant interleukin-2 (IL-2) or lymphokine-rich culture supernatant. While the effects of suboptimal concentrations of FK-506 and cyclosporin A (CsA) are additive, FK-506 does not enhance the effects of optimal concentrations of CsA. Both drugs also have similar effects on the expression of specific mRNA in Con A-activated lymphocytes. A brief preincubation of unstimulated cells with FK-506 irreversibly inhibits their subsequent responsiveness to Con A. The mechanism of action of FK-506 thus resembles that of CsA, except that it is effective at two to three orders of magnitude lower concentrations and its effects are much less readily reversible.     

Inhibition of murine B-lymphocyte proliferation by the novel immunosuppressive drug FK-506.

The novel immunosuppressive drug FK-506 inhibits both cell-mediated and humoral immunity in vivo and suppresses T-lymphocyte activation at nanamolar concentrations in vitro. We report here that FK-506 also inhibits the in vitro proliferation of murine B lymphocytes induced by goat antimouse IgM (GaMIgM), although it does not affect the induction of proliferation by bacterial lipopolysaccharide (LPS). The action of FK-506 on B-lymphocyte proliferation thus resembles that of cyclosporin A, further strengthening the correlation between the action of the two immunosuppressive drugs previously reported for T-lymphocyte activation.     

FK-506 and cyclosporin A: immunosuppressive mechanism of action and beyond.

Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.     

Effect of FK-506, a novel immunosuppressive drug on murine systemic lupus erythematosus

A novel immunosuppressive compound extracted from the fermentation broth of Streptomyces tsukubaensis belongs to the macrolide family. We gave this drug (FK-506) to MRL/lpr and NZB X NZW F1 (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE), to investigate its effect on the course of the disease. This drug showed potential to prolong the lifespan, to reduce proteinuria, and to prevent the progression of nephropathy. Appreciable differences in the levels of anti-dsDNA antibodies between treated and control animals were nil.     

Cyclosporine a and FK506 show similar immunosuppressive effects on long-term in vitro T-cell proliferation

Because Cyclosporine A (CsA) is often reported to inhibit only the proliferation of quiescent T-cells and to have much less or no effect on activated proliferating T-cells, the effects of CsA and FK506 on long-term in vitro T-cell proliferation were investigated for alloreactive T-cell clones and clones derived from a leukaemia patient early after allogeneic bone marrow transplantation. Drug effects were tested in the presence of exogenous IL2 or IL2 + IL4 during weekly T-cell activation/restimulation. In the presence of either drug, cloned T-cells were able to respond to a reduced number of restimulations (mitogenic activation, allostimulation) and could undergo fewer cell divisions/population doublings. These effects were seen in the presence of both excess IL2 or IL2 + IL4.     

T lymphocyte activation through the C28 pathway is insensitive to inhibition by the immunosuppressive drug FK-506

Nanomolar concentrations of the novel immunosuppressive drug FK-506 inhibit the proliferation of human T lymphocytes in vitro induced by mitogenic lectins or by monoclonal antibodies directed against the CD3 or CD2 surface antigens. However, the alternative pathway of T lymphocyte proliferation induced by monoclonal antibodies specific for CD28 together with phorbol esters is unaffected by FK-506.     

Spleen lymphocyte populations and expression of activation markers in rats treated with the potent new immunosuppressive agent FK-506

Rats were immunized systemically with sheep red blood cells (SRBC) and treated with either FK-506 (1 mg/kg/day) or cyclosporin A (CsA) (25 mg/kg/day) for 7 days. Profound (greater than 90%) suppression of the production of splenic IgM-secreting plasma cells and circulating antibody levels was observed in animals receiving either drug. Immunosuppression was accompanied by significant increases in the incidence and absolute numbers of OX8+ (T-cytotoxic/suppressor) lymphocytes in the spleen, and there were corresponding reductions in the W3/25+:OX8 (CD4+:CD8+) ratio. The magnitude of these changes was not affected by drug combination. There were no significant alterations in B cells with either agent, whilst a small but significant increase in the incidence of macrophages was observed in all drug-treated groups. Neither FK-506 nor CsA affected IL-2 receptor (OX39) or MHC class II (OX6) antigen expression. This study demonstrates the remarkable immunosuppressive potency of FK-506 and its underlying capacity, like CsA, to affect regulatory T-lymphocyte subsets in vivo.     

Comparative evaluation of in vitro and in vivo immunosuppressive potential of cyclosporin g with cyclosporin a and FK-506

Cyclosporin G (CsG), a promising cyclosporin A (CsA) analogue, was examined and compared with two reference immunosuppressive drugs: CsA and FK-506, regarding their inhibitory effects on different lymphocyte activation pathways as well as on graft-versus-host reaction (GvHR) across differences at major or minor histocompatibility loci. The results showed that, at different concentrations, CsG efficiently inhibited proliferation induced by alloantigens (mixed lymphocyte culture), mitogens (concanavalin A, pokeweed mitogen) and the combination of phorbol myristate acetate + ionomycin, to the same extent as observed with CsA and FK-506. It was also shown that CsG exhibited the same strong inhibitory effects as the two other immuno-suppressants upon stimulation triggered by viral (MLs-1^a) or bacterial (staphylococcal enterotoxin B) superantigen. Determination of IL-2 activity in the supernatant of MLC also confirmed similar strong inhibitory effects, exerted by CsG compared to CsA and FK-506. In systemic and local GvHR across major or minor histocompatibility barriers, CsG as well as CsA and FK-506 presented an equivalent immunosuppressive potential. In conclusion, from various experiments involving different modes of activation, it was shown that CsG was as strongly immunosuppressive as CsA and FK-506.     

Cyclosporine, FK-506 and other drugs in organ transplantation.

As experience of the most effective way to use cyclosporine for immunosuppression in organ transplantation grows, new drugs are emerging, which may improve the potency of future immunosuppressive protocols or at least provide alternative drugs in selected situations. These newer agents include FK506, which is undergoing extensive clinical trials, rapamycin, RS-61443 and deoxyspergualin. The increasing understanding of the mechanism of action of some of these drugs on signal transduction pathways in the T cell should allow the development of drugs with perhaps more specific actions.     

The influence of FK-506 on the thymus: an immunophenotypic and structural analysis.

The influence of the powerful new immunosuppressant FK-506 on the thymus was investigated in Sprague-Dawley rats that were immunized with sheep erythrocytes and treated with FK-506 (1 mg/kg/day i.m.) for 7 days. Suppression of humoral immunity in drug-treated animals was accompanied by reductions in circulating lymphocytes bearing activation markers (interleukin-2 receptor beta-chain and OX40, activated CD4+ cells) and by striking thymic medullary atrophy. There were, however, no significant differences in thymic weights or in thymocyte numbers between experimental and control groups during the period of FK-506 administration. Reduction of the medullary compartment was visualized immunohistochemically, by decreases in major histocompatibility complex (MHC) class I- and MHC class II-positive cells and in CD37+ (mature medullary) thymocytes. Flow cytometric analysis of thymocytes showed that FK-506 induced increases in bright, Thy-1.1+ cells and in numbers of CD4+ and CD8+ thymocytes, whilst CD37+ cells were less numerous than in controls. Percentages of MHC class I- and MHC class II-positive cells varied little throughout the course of FK-506 administration. Evidence of selective damage to medullary epithelial cells, attributable to FK-506, was found at both the light and electron microscopic levels, whilst thymic macrophages in drug-treated rats displayed features of enhanced phagocytic activity, including ingestion of damaged epithelial cells. These FK-506-induced abnormalities were reversed within 14 days of drug withdrawal. These findings suggest that, like cyclosporin A, FK-506 reversibly disrupts the thymic microenvironment and may interfere with the function/maturation of T lymphocytes.     

Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25-week-old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506-treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl-2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.     

The effect of the immunosuppressant FK-506 on alternate pathways of T cell activation.

Structurally unrelated, FK-506 and cyclosporin (CsA) bind to and inhibit the action of distinct cytoplasmic receptors, FK-506-binding protein (FKBP) and cyclophilin (CyP), respectively. These receptors, termed immunophilins, share no sequence similarity, and yet both have been demonstrated to be capable of catalyzing the cis-trans isomerization of peptidyl-prolyl bonds (rotamase activity). Because FK-506 and CsA bind to different intracellular target structures, we investigated the spectrum of action of FK-506, in comparison to CsA, on T cell activation. We have shown that FK-506, like CsA, is able to inhibit T cell activation mediated not only by the T cell receptor-CD3 complex, but also via another surface molecule, CD2. T cell proliferation, stimulation of interleukin 2 production, and induction of apoptosis were all sensitive to inhibition by both FK-506 and CsA. With each parameter of activation, FK-506 is approximately 10-100-fold more effective than CsA. In contrast, FK-506 did not affect T cell proliferation induced by anti-CD28 monoclonal antibody in the presence of phorbol 12-myristate 13-acetate. This CD28 pathway, however, was inhibited by a structural homology of FK-506, rapamycin, demonstrating that the mechanism of action of FK-506 has specificity. These data suggest that immunophilins or the complex of drug coupled to immunophilin (i.e. FK-506/FKBP, CsA/CyP) are involved in and regulate selective pathways of T cell stimulation.     

Antigen presentation and HLA-DR expression by FK-506-treated human monocytes.

The novel macrolide immunosuppressant FK-506 demonstrated superior potency to cyclosporin A (CsA) in the inhibition of purified protein derivative (PPD)-induced human T-cell proliferation. Pulsing of monocytes with PPD for 24 hr in the presence of FK-506 did not impair their capacity to subsequently induced the proliferation of purified autologous T cells. In contrast, FK-506 profoundly inhibited the proliferative response of T cells to antigen-pulsed monocytes. Recombinant IL-2, but not IL-1, partially restored the proliferative response to PPD in the presence of the drug. FK-506 had no effect on basal or rIFN-gamma-induced expression of HLA-DR on monocytic cell line cells. These findings provide the first evidence that FK-506 can profoundly inhibit soluble antigen-induced human T-cell proliferation at concentrations which do not significantly impair the accessory function of mononuclear phagocytes.     

Comparison of the in vitro and biophysical effects of cyclosporine A, FK-506, and mycophenolic acid on human peripheral blood lymphocytes.

The immunosuppressive drugs FK-506 and mycophenolic acid (MPA) have recently been described, but their mode(s) of action are not well understood. We have compared them to cyclosporine A (CsA) in several assays. We have shown that CsA (1 microgram/ml), MPA (0.1 microgram/ml), and FK-506 (0.5 microgram/ml) all induce a state of unresponsiveness to anti-CD3 stimulation as measured by [3H]-thymidine uptake. This suggests that the target of these drugs may be present only after mitogenic stimulation. These drugs also cause a hyperpolarization of the plasma membrane of lymphocytes. This effect is blocked by quinine or verapamil. All three immunosuppressors only slightly modulate the increase in intracellular Ca++ caused by Con-A or by anti-CD3 stimulation but do not affect Ca++ levels alone. They also decrease expression of IL-2 receptors on alpha CD3-stimulated lymphocytes. Similarities in their modes of action, as measured by these biophysical and cell biological tests, indicate the possibility that these three drugs will show similarities in their clinical performance.     

The immunosuppressant FK-506 prevents progression of diabetes in nonobese diabetic mice.

A novel immunosuppressive compound, FK-506, isolated from Streptomyces has potent immunosuppressive activities. To investigate the effect of FK-506 on the course of diabetes in nonobese diabetic (NOD) mice, we gave this drug to these animals, from the age of 8 weeks, intraperitoneally, in doses of 0.1 mg (2.5 mg/kg/day) or 0.01 mg (0.25 mg/kg/day) three times a week. Overt diabetes were observed in 75.5% of control mice by the age of 20 weeks. In contrast, no diabetes occurred in mice given 0.1 mg of FK-506. Sixteen percent of mice treated with 0.01 mg of the drug became diabetic. Administration of this drug prevented the progression of insulitis in NOD mice. The mice given 0.1 mg of FK-506 lost weight, but this was reversible.