RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.     

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.     

Differential effect of cyclosporin-A on the mixed-lymphocyte culture-induced proliferative and cytotoxic responses of T lymphocytes with different cell densities

We have analyzed the effect of cyclosporin-A (CsA) on the proliferative and cytotoxic responses induced by mixed-lymphocyte cultures (MLC-s) on low and high density T lymphocytes. Allogeneic stimulation had a different impact on the two subsets. Proliferative and cytotoxic responses were inversely correlated; i.e., high density cells proliferated but exerted low levels of cytotoxicity while the lytic activity of the low density subset was stronger and the proliferation was weak. CsA impaired the proliferative and cytotoxic responses of the high density T lymphocytes but influenced less markedly the response of the low density cells. In both subsets CsA inhibited the MLC-induced interleukin 2 (IL-2) production. The generation of specific cytotoxicity was markedly suppressed by CsA, whereas the generation of anomalous activity was less affected. Addition of exogenous IL-2 to the CsA-containing cultures fully restored the proliferation and the generation of nonspecific cytotoxicity. In contrast, addition of interferon gamma (IFN-gamma) restored neither of these responses. However, for complete restoration of the stimulation-specific cytotoxicity, addition of both lymphokines was required. Taken together these results suggest that the CsA-induced suppression of the lymphokine production has different consequences in the low and high density subsets; the expression of anomalous and specific cytotoxicities require different signals; CsA interferes with several steps in the T-cell activation.     

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.     

Differential effects of cyclosporin A on diverse B cell activation phenomena triggered by crosslinking of membrane IgM.

Cyclosporin A (CsA) was tested for its modulatory effects on the mIgM-mediated signaling of G0*-associated increases in class II MHC expression, G1-related RNA synthesis, and S phase-related DNA synthesis in human B cells. While CsA at concentrations as low as 10-100 ng/ml could completely ablate anti-IgM-induced DNA synthesis, earlier G1-associated RNA synthesis was only partially inhibited, and signaling of increased membrane class II MHC expression was unaffected by up to 1000 ng/ml of CsA. Similar phenomena were observed in a clonal population of leukemic B lymphocytes susceptible to anti-IgM-mediated activation in the absence of T cells and T cell factors indicating (a) that the inhibitory effects are not due to CsA-mediated suppression of cytokine production by contaminating T cells, and (b) that the varying effects of CsA on the diverse activation phenomena do not reflect B cell subpopulation diversity. Pulsing studies revealed that while maximal suppression of anti-IgM-induced G1-associated RNA synthesis required CsA at culture initiation, near maximal suppression of DNA synthesis occurred when CsA, or soluble human IgM, was added up to 30 hr after the initial exposure of resting B cells to the anti-IgM ligand. These latter findings are consistent with the possibility that the CsA-mediated suppression of S phase entry is due to the inhibition of a signaling event proximal to mIgM ligation which must be repeatedly initiated throughout the first 30 hr of activation.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.     

Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25)

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+-sensitive dye Indo-1 showed that concentrations of CsA sufficient to inhibit 90-100% of tritiated thymidine incorporation had no effect on the PHA-stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37 degrees C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin-2, IL-2), as measured by monoclonal antibodies to CD25 after 16-24-hr incubation. In like manner, CsA also prevented the increase in the expression of the E-rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40-72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of "activation antigens."     

Immunosuppression in human peripheral blood T lymphocytes by fluvastatin

Statins are competitive inhibitors of HMG-CoAreductase, which is the major rate-limiting enzyme thatcontrols the conversion of HMG-CoA to mevalonic acid[1]. Mevalonate derived intermediates, such as isoprenoid,farnyesylpyrophosphate and geranylpyrophosphate, serveas important lipid attachments for the posttranslationalmodification of a variety of proteins such as small GTP-binding proteins of the Ras and Rho superfamily involvedin intracellular signaling [2]. Therefore, apart from the we…     

IL-4 bypasses the immune suppressive effect of cyclosporin A during the in vitro induction of murine cytotoxic T lymphocytes

We have analyzed at the clonal level the effect of IL-4 on the immune suppressive action of cyclosporin A (CsA) during the in vitro primary activation of anti-MHC alloantigen-reactive murine CD8+ CTL. Although neither IL-4 nor IL-2 alone were able to overcome the CsA-mediated suppression, the addition of IL-4 in the presence of IL-2 restored in a dose-dependent manner the induction of cytolytic activity. On the other hand, CsA greatly impaired proliferative responses of alloantigen-reactive CD8 T cells, thereby operationally dissecting proliferative responsiveness from acquisition of cytolytic activity during primary activation of alloantigen-reactive CD8+ T cells. The existence of a CsA-resistant induction pathway for Ag-specific CD8+ T cell-mediated cytolytic activity may be of relevance for experimental and clinical organ transplantation.