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.     

FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes.

This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation

The present study shows the in vitro effects of a novel immunosuppressive agent, FK506, in comparison with cyclosporin A (CsA). FK506 inhibited concanavalin A response and allo-mixed lymphocyte reaction of murine splenic lymphocytes in a dose-dependent manner, and at 40- to 200-fold lower concentrations than CsA. Allo-cytolytic T lymphocyte induction from murine thymocytes was also inhibited by FK506, whereas the ability of cytolytic T lymphocyte to lyse targets was not affected by the agent. Immunosuppressive effects of FK506 were further characterized by using antigen specific-proliferative T lymphocyte clones, BC.21 and KO.6. FK506 inhibited the proliferation of T cell clones stimulated with specific antigens in a dose-dependent manner, and at about 100-fold lower concentrations than CsA. However, cloned T cells, once activated, were scarcely affected by the agent; interleukin-2 (IL-2) driven proliferation of cloned T cells was not inhibited. On the other hand, it was found that FK506 inhibited both IL-2 secretion and IL-2 receptor expression of BC.21 after stimulation with the specific antigen. FK506 also inhibited the proliferation of BC.21 stimulated with phorbol 12-myristate 13-acetate plus calcium ionophore, indicating that it directly affected the signaling pathway downward from the perturbation of the Ti/T3 complex. Finally, it was suggested that FK506 and CsA synergistically inhibited the antigen-driven proliferation of cloned T cells. These results indicate that the novel immunosuppressive agent, FK506, affects T cell activation with mechanisms similar to those of CsA but at considerably lower concentrations.     

Local regulation of immune responses: corneal endothelial cells alter t cell activation and cytokine production

Corneal endothelial cells (CE cells) inhibit antigen- and mitogen-activated lymphocyte proliferation assays, although interleukin 2 receptor (IL-2R) expression and responsiveness to exogenous IL-2 are unaffected. To examine this activity further, co-cultures of CE cells and T cell clones were studied. CE cells inhibited IL-2 and IL-4 production by T cells stimulated with Ag and APC, but not IL-5 or IL-6 production. CE cells also inhibited NFAT-driven lacZ reporter gene production following Ag stimulation of transfected KZO T hybridoma cells. Conversely, stimulation of IL-2 production by ionomycin, with or without PMA, was unaffected by the CE cells. Preincubation of KZO hybridoma or Jurkat cells with CE cells, or CE cell-conditioned culture supernatant, inhibited the intracellular calcium ([Ca(2+)](i)) increase induced by TCR ligation, but not the [Ca(2+)](i)increase induced by ionomycin or thapsigargin. The inhibitory effect was independent of APC and did not act by blocking costimulation, since IL-2 production stimulated by immobilized anti-CD3 alone was also inhibited by CE cells. The supernatant factor was heat labile. This novel activity is unlike other immunoregulatory molecules, including transforming growth factor beta (TGF-beta) and may contribute to local immune privilege.     

Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Cyclosporin A inhibits initiation but not progression of human T cell proliferation triggered by phorbol esters and calcium ionophores

Cyclosporin A (CsA) is a potent inhibitor of T lymphocyte proliferation induced by Ag and mitogens. In an attempt to further delineate the mechanism of action of CsA, we have examined its effects on T cell proliferation induced by the combination of the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin. T cells were rendered competent as the result of a 30-min initial incubation with both drugs, after which the drugs were washed out. Competence is defined as the ability to subsequently proliferate in response to exogenously added IL-2 or PDB in the second phase of the culture, but not to synthesize IL-2 or proliferate without these additions. Addition of CsA (1 microgram/ml) to the cells in the initial, competence-inducing 30-min incubation with PDB/ionomycin abrogated their subsequent response to IL-2 or PDB. In contrast, addition of CsA to cells after they had been treated for 30 min with PDB/ionomycin and then washed did not affect their responses to subsequent addition of either IL-2 or PDB. Treatment with CsA during induction of competence prevented the expression of the 55-kDa IL-2R gene during competence induction and inhibited IL-2 gene expression and IL-2 production in response to PDB in the second phase. These results indicate that the effects of CsA are limited to the initiation (competence induction) period of T cell activation, that CsA apparently affects expression of more than one gene, and in competent cells, CsA does not affect their ability to progress to DNA synthesis.     

FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein, cyclophilin

A novel macrolide antibiotic, FK-506, isolated from Streptomyces tsukubaensis, has been shown to be a potent immunosuppressive agent in vivo and in vitro. FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporin A (CsA), although 10 to 100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism(s) of action at the biochemical level. We have identified a cytoplasmic binding protein for FK-506 in the human T cell line, JURKAT, using [3H]FK-506. The FK-506 binding protein has a mr of 10 to 12 kDa (as determined by gel filtration), is heat stable and does not bind CsA. This contrasts with the CsA binding protein, cyclophilin, in that cyclophilin is heat labile and has a mr of 15 to 17 kDa. Our data suggest that FK-506 binds to a low m.w. protein(s) in JURKAT cells, which is distinct from cyclophilin. This protein may mediate the immunosuppressive effects of FK-506 in T cells. In addition, our results suggest that the immunosuppressive activity of FK-506, as with CsA, is mediated by an intracellular mechanism.     

Signal transduction pathways that contribute to increased protein synthesis during T-cell activation

Protein synthesis rates were maximally stimulated in human lymphocytes by ionomycin and the phorbol ester PMA (I+P), which promotes proliferation, whereas PMA alone, which does not promote proliferation, stimulated protein synthesis to a lesser degree. Three translation-associated activities, eIF4E phosphorylation, eIF2B activity and 4E-BP1 phosphorylation also increased with stimulation by I+P and PMA, but only 4E-BP1 phosphorylation was differentially stimulated by these conditions. Correspondingly, signaling pathways activated in T cells were probed for their connection to these activities. Immunosuppressants FK506 and rapamycin partially blocked the protein synthesis rate increases by I+P stimulation. FK506 had less of an inhibitory effect with PMA stimulation suggesting that its mechanism mostly affected ionomycin-activated signals. I+P and PMA equally stimulated phosphorylation of ERK1/2, but I+P more strongly stimulated Akt, and p70(S6K) phosphorylation. An inhibitor that blocks ERK1/2 phosphorylation only slightly reduced protein synthesis rates stimulated by I+P or PMA, but greatly reduced eIF4E phosphorylation and eIF2B activity. In contrast, inhibitors of the PI-3 kinase and mTOR pathways strongly blocked early protein synthesis rate stimulated by I+P and PMA and also blocked 4E-BP1 phosphorylation and release of eIF4E suggesting that these pathways regulate protein synthesis activities, which are important for proliferation in T cells.     

Modulation of the electrophoretic mobility of the linker for activation of T cells (LAT) by the calcineurin inhibitors CsA and FK506: LAT is a potential substrate for PKC and calcineurin signaling pathways

The linker for activation of T cells (LAT) is essential for T cell activation. Cyclosporin A (CsA) and FK506, inhibitors of T cell proliferation, have been very useful for preventing autoimmune and inflammatory disease and graft rejection. However, both compounds are associated with side effects. We show that TCR ligation in the presence of FK506 or CsA induced rapid modifications in LAT that modulate the electrophoretic mobility of the molecule in SDS-PAGE. Calcineurin, a target for CsA and FK506, dephosphorylated LAT in vitro and restored its electrophoretic mobility. Stimulating T cells with the protein kinase C (PKC) activator PMA induced a shift in the mobility of LAT, whereas inhibitors of PKC blocked the effect of PMA. Thus, manipulating calcineurin or PKC activation alters the electrophoretic mobility of LAT. These results shed light on the molecular actions of CsA and FK506 in T cells and implicate LAT in mediating the drugs' actions.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Demonstration of a direct inhibitory effect of cyclosporine on normal human T-cells with two novel models of T-cell activation as probes

Two novel models of activation of human peripheral blood quiescent T-cells (T-cells) were utilized herein as probes to analyze the mechanisms and to locate the site of action of cyclosporine (CsA) in the T-cell activation pathway. Highly purified T-cells were activated, independently of accessory cells, with either crosslinked anti-CD2 + anti-CD3 monoclonal antibodies (mAbs) or with sn-1,2-dioctanoylglycerol (DAG) and ionomycin. CsA inhibited the expression of 55-kDa interleukin-2 receptors (IL-2R) and T-cell proliferation in these accessory cell-independent models of T-cell activation. Recombinant IL-2, over a wide range of concentrations that included different binding affinities of cellular receptors for IL-2, did not completely reverse CsA-associated inhibition of IL-2R expression and/or proliferation. In additional experiments, designed to examine early activation related events, CsA did not interfere with the increase in intracellular free calcium concentration initiated with anti-CD2, anti-CD3, anti-CD2 + anti-CD3 mAbs or with ionomycin. DAG-induced and PKC-activation-dependent down-regulation of cell surface expression of CD3 antigens was similarly unaffected by CsA. Our findings unambiguously indicate that CsA has a direct inhibitory effect on T-cells. Moreover, CsA's cellular site of action is distal to calcium mobilization and PKC activation but proximal to IL-2R expression and IL-2-dependent DNA synthesis in normal human T-cells.     

用于靶向于NFAT的免疫抑制类小分子化合物筛选的细胞模型的建立

建立IL-2启动子以及NFAT-AP1增强子调控报告基因包括D2egfp或Luciferase在Jurkat细胞中瞬时表达的细胞模型。首先,利用三步连接将IL-2-promoter -255～+285处的序列插入到Pnf-Κb-D2egfp表达载体启动子上游,形成3个拷贝的前后串联的增强子序列;然后从人外周血钓取两条IL-2-promoter序列,包括-326～＋46以及-89～＋46两段基因组序列,分别替代上述重组表达载体中的TK minimal promoter, 构建所需的IL2 promoter以及NFAT-AP1 enhancer调控的报告基因表达质粒：3×NFAT-AP1-IL-2P/D2egfp和3×NFAT-AP1-TATA/D2egfp; 最后,分别将3×NFAT-AP1-IL-2P以及3×NFAT-AP1-TATA融合基因克隆到Pgl3-basic载体中,构建相应的Luciferase报告基因表达质粒。利用电转染方法将重组的报告基因表达载体瞬时转入Jurkat细胞后发现,未刺激以及PMA、离子霉素（Ionomycin）单刺激均不能激活下游报告基因的表达,只有PMA和离子霉素联合刺激才能启动D2egfp以及Luciferase的转录表达,并且5μg/Ml FK506预先作用1h能几乎完全阻断刺激剂诱导的无论是IL-2 promoter还是NFAT-AP1enhancer调控的报告基因的表达。实验结果提示,所构建的Jurkat细胞瞬时表达模型可用于靶向于NFAT信号通路的FK506类免疫抑制小分子化合物的初步筛选。