Changes in the redox status of cells affect NF-kappa B and activator protein (AP)-1 nuclear expression and activity. In particular antioxidants decrease NF-kappa B and increase AP-I transcriptional activity, thereby regulating gene expression. In T cells, low concentrations of antioxidants enhance IL-2 and inhibit IL-4 expression. Since NFAT binding sites play an essential role in regulating IL-2 and IL-4 gene transcription, we studied the effects of dithiocarbamates, using the pyrrolidine derivative of dithiocarbamate (PDTC), on the activity of the distinct AP-I-containing IL-2 NFAT and AP-I-less IL-4 NFAT enhancers elements. Consistent with the presence of AP-I proteins within the IL-2 NFAT complex, PDTC strongly enhanced phorbol 12-myristate 13-acetate/phytohemagglutinin-induced NFAT binding to the IL-2 NFAT enhancer and transcriptional activity of a reporter plasmid driven by this NFAT enhancer. In contrast, the activity of the IL-4 NFp enhancer, which does not bind AP-I, was abolished by PDTC treatment. In the Jurkat T cell line treated with PDTC, cc-expression of the Ca2+/calmodulin-dependent phosphatase, calcineurin, completely restored the IL-4 NFp enhancer activity. Our data indicate that calcineurin-mediated NFAT activity is a target for antioxidants and provides new insights into the molecular mechanisms controlling differential cytokine gene expression.

ABSTRACT Advanced stages of mycobacterial diseases such as leprosy and tuberculosis are characterized by a loss of T-cell function. The basis of this T-cell dysfunction is not well understood. The present report demonstrates major alterations in the expression of signal transduction molecules in T cells of leprosy patients. These alterations were most frequently observed in lepromatous leprosy (LL) patients. Of 29 LL patients, 69% had decreased T-cell receptor ζ-chain expression, 48% had decreased p56lck tyrosine kinase, and 63% had a loss of nuclear transcription factor NF-κB p65. An electrophoretic mobility shift assay with the gamma interferon core promoter region revealed a loss of the Th1 DNA-binding pattern in LL patients. In contrast, tuberculoid leprosy patients had only minor signal transduction alterations. These novel findings might improve our understanding of the T-cell dysfunction observed in leprosy and other infectious diseases and consequently might lead to better immunologic evaluation of patients.

Interleukin (IL)‐4 is an immunomodulatory cytokine produced by a number of cell types including T cells, basophils, and mast cells. This pleiotropic cytokine has a number of immunoregulatory functions; however, the molecular mechanisms controlling the transcription of this gene are not yet completely understood. Several studies have implicated a possible autoregulatory mechanism for its own expression. Here, we have identified a Stat‐6‐responsive element (Stat‐6RE) in the promoter of the human IL‐4 gene. Utilizing electrophoretic mobility shift analysis, we have demonstrated the presence of two specific IL‐4‐responsive DNA‐protein complexes in nuclear extracts of both human Th1 and Th2 clones. Phytohemagglutinin‐blasted peripheral blood T cells also generated an inducible complex in response to stimulation with IL‐4 and the IL‐4‐like cytokine IL‐13. Transient transfection of the murine pre‐B cell line BA/F3 stably transfected with the full‐length human IL‐4 receptor α chain demonstrated the ability of multicopy Stat‐6RE to initiate transcription from a heterologous promoter upon IL‐4 or IL‐13 stimulation. These results indicate a possible autocrine mechanism for the regulation of IL‐4 gene transcription through the Stat‐6RE as well as a possible mechanism for IL‐13 regulation of the human IL‐4 promoter.

The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 delta epsilon gamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain.

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappa B protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappa B consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappa B site (IFN-gamma kappa B) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappa B and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappa B subunits bind specifically to the newly identified IFN-gamma kappa B and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappa B proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappa B and the C3-3P site to enhance gene transcription.These results demonstrate that the coordinate activities of NFAT and NF-kappa B proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.

The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.

Disregulation of vitamin A metabolism is able to generate different immunological effects, including altered response to infection, reduced IgG production, and differential regulation of cytokine gene expression (including interleukin-2 and -4 and interferon-gamma (IFN-gamma)). In particular, IFN-gamma gene expression is significantly affected by vitamin A and/or its derivatives (e.g. retinoic acid (RA)), Here, we analyze the effect of retinoic acid on IFN-gamma transcription. Transient transfection assays in the human T lymphoblastoid cell line Jurkat demonstrated that the activation of the IFN-gamma promoter was significantly down-regulated in the presence of RA, Surprisingly, two different AP-1/CREB-ATF-binding elements situated in the initial 108 base pairs of the IFN-gamma promoter and previously shown to be critical for tran scriptional activity were unaffected by Rk Utilizing promoter deletions and electrophoretic mobility shift analysis, we identified a USF/EGR-1-binding element cooperating in the modulation of IFN-gamma promoter activity by RA, This element was found to be situated in a position of the IFN-gamma promoter close to a silencer element previously identified in our laboratory, These results suggest that direct modulation of IFN-gamma promoter activity is one of the possible mechanisms involved in the inhibitory effect of retinoids on IFN-gamma gene expression.

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.

Flavone-8-acetic acid plus recombinant human interleukin 2 is a successful antitumor therapy in mice bearing the Renca murine renal cell carcinoma. This report demonstrates that T cells, particularly CD8+ T cells, are critical for the generation of this response. Initial experiments examining T-cell signal transduction proteins demonstrated that T cells from Renca-bearing mice had undetectable levels of p56lck and zeta-chain of the T-cell receptor and that flavone-8-acetic acid and recombinant human interleukin 2 therapy could be used as a model for reversal of these alterations. However, further experimentation showed that the majority of the reduction in zeta-chain and part of the reduction in p56lck was due to degradation of these molecules during protein extraction caused by mature granulocytes contaminating the enriched T-cell population. This was not the case for nuclear c-Rel or NF kappa B p65, which remained at undetectable/reduced levels in the absence of granulocytes, confirming our previous data that transcription factor alterations exist in tumor-bearing mice. Thus, most of the reduction in zeta-chain in T cells from Renca-bearing mice is due to granulocyte contamination and emphasizes the need to use pure T-cell populations and/or sufficient amounts and types of protease inhibitors when quantitating proteins in T cells from tumor-bearing mice.

BACKGROUND:The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by T helper 1 (Th1) and T helper 2 (Th2) T-cell subsets, which are two arms of the immune system. Th1 cells preferentially produce interleukin 2 (IL-2), interferon gamma (IFN gamma), and tumor necrosis factor (TNF), resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and stimulate an antibody response that helps to prevent the cells from becoming infected. The clinical progression of several infectious diseases, including human immunodeficiency virus, some types of parasitoses, and tuberculosis, is thought to be associated with the predominance of a Th2-type T-cell response. Recent reports have demonstrated the presence of T cells producing Th2 lymphokines (IL-4, IL-6, and IL-10) in tumor-infiltrating lymphocytes of renal cell carcinoma.PURPOSE:The purpose of this study was to investigate at the molecular level whether there was any change in the splenic T cells of mice with progressively growing tumors from a Th1 to a Th2 DNA-binding pattern or phenotype.METHODS:Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma were tested for cytokine production after in vitro activation. Nuclear extracts of splenic T cells were used for the DNA-binding assay using IFN-gamma core promoter region, the kappa B (kappa B) site from immunoglobulin gene, and the nuclear factor of activated T-cell (NFAT) site from IL-2 gene.RESULTS:Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma preferentially produced Th2 cytokines (i.e., IL-4) upon activation and showed a marked decrease in Th1 cytokine (particularly IFN gamma) production compared with the production observed in normal splenic T cells. The DNA-binding assay with the IFN-gamma core promoter region confirmed the gradual decline in the nuclear transcription factors associated with the Th1 phenotype during tumor progression in both tumor models. Renal cell carcinoma-bearing mice, successfully treated with flavone-8-acetic acid and recombinant human IL-2, showed a reversion to a Th1-like pattern. In addition, nuclear extracts of T cells from tumor-bearing animals showed a Th2-type kappa B-binding pattern. Moreover, the NFAT complex present in the normal splenic T cells was lost at the later stages of tumor progression; instead, a new complex was present in mice bearing long-term tumors.CONCLUSION:T cells from tumor-bearing mice lose the Th1 phenotype with progressive tumor growth.

Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 . CREB-ATF factors were able to block the glucocorticoid effect.These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 . CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells.

Previous studies have suggested that gangliosides have an important role in cell signaling and recognition. However, their specific function in these processes has not been clearly defined. A mAb, R24, that reacts specifically with a cell surface ganglioside (GD3) has been demonstrated to stimulate proliferation of T cells derived from human peripheral blood. In this study, we have investigated the mechanisms by which the R24 mAb affects T cell functions. We have observed that the R24 mAb stimulates GD3+ T cell proliferation, cytotoxicity, and surface marker expression of IL-2R alpha-chain, IL-2R beta-chain, HLA-DR, CD11a, and CD11c. Additionally, IFN-gamma activity but not IL-1, IL-2, or IL-4 activity was present in culture supernatants 72 h after R24 stimulation. In some donors, increased IL-6 and TNF-alpha activity also was detected after R24 treatment. Furthermore, R24 treatment resulted in translocation of c-rel, but little or no NFkappaB p50 or p65, from the cytoplasm to the nucleus and an increase of NFkappaB binding complexes containing c-rel and p50. This treatment also caused increased tyrosine phosphorylation of specific protein substrates. R24-stimulated increases in proliferation, cytotoxicity, and cell surface protein expression could be blocked by cyclosporin and staurosporin, indicating that cyclophilin/calcineurin and protein kinase C may be involved in the R24 signaling pathway. Additionally, herbimycin A, a tyrosine kinase inhibitor, blocked the R24-stimulated increase in proliferation but not cytotoxicity at concentrations consistent with specificity for tyrosine kinases. These results suggest that multiple biochemical pathways are involved in the activation of human T cells by R24.

Previous analysis of the human interferon-gamma (IFN-gamma promoter indicated that the region of DNA from -251 to -215 (designated here as BE (binding element)) possessed silencer activity, as deletion of this region caused an increase in promoter activity. Based on this finding, we have conducted a series of experiments to characterize BE function and analyze the binding proteins which interact with this region. Transient transfection assays in the Jurkat T cell line revealed that the BE region possesses silencer activity, which is orientation-dependent when reinserted 5' to the IFN-gamma core promoter. However, when the BE region was inserted in front of a heterologous promoter (thymidine kinase (TK)), a mild enhancer activity was observed. Utilizing the electrophoretic mobility shift assay, we have identified two major DNA-protein complexes (designated as S and E complexes) which interact with this region. Mutational analysis indicated that the silencer activity observed with the IFN-gamma promoter correlated with the S complex and the enhancer activity correlated with the E complex. Preliminary characterization of these two DNA-protein complexes has demonstrated the presence of multiple proteins in each complex. We have found that the S protein complex has a recognition sequence similar to the nuclear factor AP2, and we have identified the nuclear factor Yin-Yang 1 (YY1) as one of the proteins in the E complex.

It has recently been shown that T-cell signal transduction molecules are altered in tumor-bearing mice. We have examined the expression of NF kappa B/Rel family proteins in T-cells from mice bearing Renca, a murine renal carcinoma. T-cells from Renca-bearing mice expressed undetectable levels of nuclear c-Rel, NF kappa B p65, and p50; however, two shorter forms of p50 (p48 and p46), truncated at the NH2-terminus, were present exclusively in the nucleus and were able to bind DNA. These T-cells have reduced expression of gamma-interferon mRNA. In mice successfully treated with flavone 8-acetic acid and recombinant human interleukin 2, the T-cells expressed normal levels of all three nuclear NF kappa B/Rel proteins. These results suggest that alterations in transcription factors may accompany changes in signal transduction molecules in T-cells from tumor-bearing animals; however, the changes are reversed with successful biological therapy.

Young, H. A.; Norihisa, Y.; McVicar, D.; Ghosh, P.; Houghton, A. N.; Longo, D. L.; Creekmore, S.; Ortaldo, J. R. Author Information