Prolactin expression is induced in Jurkat T-cells by beta-catenin LEF-1, AP-1 and cAMP.

Prolactin (PRL) in humans is produced in the pituitary as well as in extra-pituitary sites. A proximal promoter that requires the Pit-1 transcription factor controls pituitary PRL expression, whereas a distal (upstream) promoter located at 5.8 kb upstream of the pituitary start site regulates extra-pituitary PRL synthesis. We have previously reported that cAMP regulates PRL transcription in Jurkat lymphocytes in part through a cAMP responsive element. Here we demonstrate that additional PRL regulatory elements corresponding to LEF-l and AP-1 transcription factor binding sites appear important for PRL expression, since factor binding by EMSA and reporter gene expression are reduced when these sites are deleted or mutated. Interestingly, over-expression of a constitutively active form of beta-catenin increases PRL expression of Jurkat cells. This effect occurs through both LEF-dependent and -independent pathways. Our studies identify the distal PRL promoter as a target for beta-catenin, and reveal novel pathways regulating extra-pituitary PRL expression.

The human prolactin gene upstream promoter is regulated in lymphoid cells by activators of T-cells and by cAMP.

Prolactin (PRL) is produced in human thymocytes, T-cells and endometrium. In these extrapituitary tissues, PRL gene transcription is directed by an alternative upstream promoter, and it is thought to act as a locally produced cytokine, with relevance for immune regulation and modulation of T-cell function. We have studied PRL transcriptional regulation in the human T-lymphoblastoid Jurkat cell line transfected with a fragment of the upstream promoter linked to luciferase. A cAMP analogue (cptAMP) increased promoter activity rapidly and dose dependently. This increase was resistant to inhibition by cyclosporin A and thus independent of calcineurin phosphatase (CN). T-cell activation by phorbol myristate acetate (PMA) failed to enhance promoter activity but phytohaemagglutinin (PHA) alone or PHA+PMA increased it, and cptAMP acted in synergy with PMA or PHA to increase it further. H-89, a cAMP-dependent protein kinase A (PKA) inhibitor, inhibited the effect of cptAMP as did transfection with protein kinase inhibitor PKI, an expression vector of the specific inhibitor of PKA. A single point mutation in the CRE (cAMP response element) located at -25 bp in the PRL upstream promoter (TGACGT to TGCCGT) failed to reduce the response to cptAMP, while mutations or deletion of four nucleotides in the CRE to TACTCT diminished the response to cAMP by more than half. We conclude that activity of the human PRL upstream extrapituitary promoter can be induced by activators of T-cells, as well as by a cAMP analogue. The signal is transmitted by PKA and the effect of cAMP is independent of CN. It is partly dependent on an intact proximal CRE motif but a more upstream enhancer may contribute to promoter regulation.

Activation of human thymocytes induces the phosphorylation of protein tyrosine phosphatase 1C.

Phosphorylation of docking proteins is essential for signal transduction. In this report we provide evidence that activation of human thymocytes in culture induces the phosphorylation of the protein tyrosine phosphatase 1C (PTP 1C). Thymocytes were activated with Con A, PMA or Con A+PMA. The enzyme is phosphorylated on its serine and threonine residues. Phosphorylation occurs within 5 min and lasts for 24 h. PTP 1C is phosphorylated by PKC in vivo and in vitro; however, phosphopeptide mapping suggests that in addition to PKC other kinases phosphorylate the enzyme. On the maps of tryptic digests of cultured thymocytes more radiolabeled phosphopeptides are visualized than on the maps of digests of PTP 1C phosphorylated with partially purified PKC alpha or beta. Phosphorylation of PTP 1C decreases its activity, whereas dephosphorylation increases its activity, suggesting that phosphorylation of PTP 1C takes part in the regulation of signal transduction.

Comparison of the properties of the CsA analogs monoacetyl CyC (o-acetyl-threonine2 cyclosporin) and methyl-alanyl CsA (N-methyl-L-alanyl6 cyclosporin); monoacetyl cyclosporin is immunosuppressive without binding to cyclophilin.

Cyclosporin (CsA) is an immunosuppressant which binds to cyclophilin (Cyp). The relationship between Cyp binding and immunosuppression has been questioned since one of the analogs of CsA, N-methyl-L-alanyl6 cyclosporin (methyl-alanyl CsA) binds to Cyp but is not immunosuppressive. We compared the immunosuppressive properties of CsA, methyl-alanyl CsA and o-acetyl-threonine2 cyclosporin (monoacetyl CyC), since monoacetyl CyC does not bind to Cyp when tested in cell-free assays and its immunosuppressive properties had not been tested. Cyp is a peptidyl-prolyl isomerase which is abundant in all human tissues, yet the activities of CsA are mostly confined to inhibition of T cell and thymocyte activation, and to neuro- and nephro-toxicity and are independent of inhibition of the isomerase. Activation of thymocytes and of T cells is regulated by the binding of a nuclear factor(s) (NFs) to the NF-AT region (-285 to -255) of the IL-2 promoter. We studied inhibition of binding to the NF-AT region of NFs derived from primary cultures of thymocytes treated with CsA or its analogs. In addition, we compared the effect of CsA and its analogs on the expression of the IL-2 gene in a stably transfected Jurkat-cell line (Fgl 5) which contains three copies of NF-AT and the reporter enzyme beta-galactosidase; and on inhibition of proliferation induced by concanavalin A (Con A) or IL-2. We found that monoacetyl CyC which does not bind to Cyp is immunosuppressive by our criteria when tested in cultured cells due to either a different mechanism of action or to metabolic activation.

Molecular mode of action of cyclosporin and FK506 in human thymocytes.

The molecular mode of action of cyclosporin, three of its non-immunosuppressive analogs, N-methyl-l-alanyl cyclosporin, acetyl cyclosporin and cyclosporin S3, and of FK506 was studied in primary cultures of human thymocytes. Nuclear factors derived from thymocytes activated with phorbol myristate acetate and concanavalin A were tested for their ability to bind to a synthetic radiolabelled probe corresponding to the NF-AT region (-285 to -255) of the IL-2 gene. Binding was observed, and it was inhibited by CsA (100 ng/ml), while the analogs at ten-fold higher concentrations (1000 ng/ml) were only partially inhibitory. CsA in combination with FK506 inhibited binding of nuclear factors at the NF-AT site, and acted in concert.

Regulation of IL-2 beta receptor expression and beta-chain mRNA by human thymocytes.

The high affinity form of the human IL-2 receptor (IL-2R) has two known components, the IL-2R alpha (p55) and the IL-2R beta chain (p75). We have previously shown that recombinant IL-2 (rIL-2) could induce the expression of the alpha-chain (p55) on T cells and thymocytes, and increase this expression following suboptimal activation with concanavalin A (Con A) in combination with IL-2. An increase in the accumulation of IL-2R alpha-specific mRNA induced by rIL-2 in T cells and thymocytes had also been documented. We report here that the expression of IL-2R beta on the cell surface can be demonstrated on human thymocytes by the binding of Mik beta1, a MoAb directed against an epitope of the beta-chain. The IL-2R beta chain is constitutively expressed on freshly isolated thymocytes; this expression can be increased in thymocytes activated with Con A in combination with IL-2 or tetradecanoylphorbol 13-acetate (TPA). Blocking the formation of high affinity receptors with a MoAb directed against the alpha-chain of the receptor results in an increase in the display of IL-2R beta as evidenced by binding of MoAb Mik beta1. The accumulation of IL-2R-beta-specific mRNA is observed in freshly isolated thymocytes and it is increased in thymocytes cultured with rIL-2 alone, with Con A, and further enhanced by the addition of rIL-2 in combination with Con A or with TPA. Cyclosporine (CsA), which inhibits the accumulation of lymphokine-specific mRNA of thymocytes, does not inhibit the induction of the accumulation of IL-2R beta-specific mRNA. This is analogous to its effect on the expression of the alpha-chain (p55), and the accumulation of alpha-chain-specific mRNA.

Inhibition by chlorpromazine of lymphokine-specific mRNA expression in human thymocytes.

This study was designed to determine the effect of the phenothiazine chlorpromazine (CPZ) on the activation of human thymocytes. We provide evidence that CPZ inhibits the accumulation of mRNA specific for the lymphokines, interleukin 2, interferon-gamma, tumor necrosis factor alpha and the proto-oncogene c-myc; by contrast, the accumulation of mRNA specific for the alpha chain of the interleukin 2 receptor and the subsequent early expression of Tac antigen on the cell surface is not inhibited by CPZ. The inhibition of the expression of lymphokine-specific mRNA results in a decrease in interferon-gamma synthesis and in inhibition of thymocyte proliferation as determined by the incorporation of [3H]thymidine. In addition, we show that activation of protein kinase C (PKC) in human thymocytes by 12-O-tetradecanoyl phorbol 13-acetate (TPA) causes the phosphorylation of a protein of a molecular mass of approximately 75 kDa. The function of this protein is as yet not defined, but it is possible that it plays a role in the transduction of the signals to the nucleus which in turn elicit the expression of the genes coding for c-myc and for the lymphokines required for thymocyte activation. We also demonstrate that CPZ, like the immunosuppressant drug cyclosporin A does not inhibit the phosphorylation of the 75-kDa protein which is induced by the activation of PKC by TPA and does not affect phosphoinositide breakdown, indicating that it exerts its effect at a site distal to the activation of PKC. These observations demonstrate that CPZ has an immunoregulatory function in addition to its psychotropic activity.

Immunoregulation and production of tumor necrosis factor alpha by human thymocytes.

We have provided evidence that tumor necrosis factor alpha (TNF-alpha) enhances the proliferation and the state of activation of human thymocytes cultured with concanavalin A or interleukin 2 (IL-2), as evidenced by an increase in the expression of the c-myc gene and the gene of the IL-2 receptor (alpha-chain, Tac antigen) and by the expression of Tac antigen on the cell surface. Our observations suggest that TNF-alpha interacts with IL-2 and with another factor(s) which is induced in the course of activation by concanavalin A, since the immunosuppressant drug cyclosporin A-, which inhibits thymocyte activation, prevents the effect of TNF-alpha on thymocytes activated with concanavalin A, whereas anti-Tac, which prevents the binding of IL-2 to its receptor without affecting the production of IL-2 or the expression of IL-2-specific mRNA, inhibits proliferation only partially. By contrast, anti-Tac inhibits the response to TNF-alpha of thymocytes induced with IL-2 completely. These observations show that TNF-alpha exerts a potentially important immunoregulatory effect in synergy with IL-2 on thymocytes, which could contribute to tumor rejection. In addition, we show that activated human thymocytes express the TNF-alpha gene and that the expression of this gene is inhibited by cyclosporin A and dexamethasone.

Lymphokine synthesis is induced in human thymocytes by activation of the CD 2 (T11) pathway.

This study shows that unfractionated thymocytes can be activated to proliferate in response to activation by the CD 2 pathway, to express interleukin 2 receptors, and to synthesize interleukin 2 and interferon-gamma. Less mature, T3- thymocytes, isolated by negative selection are activated to a lesser extent than are unfractionated thymocytes; activation by the CD 2 pathway, induces proliferation, the expression of the interleukin 2 gene and interferon-gamma synthesis. 12-O-Tetradecanoyl phorbol 13-acetate in combination with the anti-CD 2 antibodies T11(2) + T11(3) increases the response of both unfractionated and T3- thymocytes. In addition we demonstrate that tetradecanoyl phorbol acetate in combination with T11(3) can replace the requirement for T11(2) for thymocyte activation and induce the expression of T11(3).

c-myc gene expression and activation of human thymocytes.

The relationship between c-myc expression and thymocyte activation was studied in freshly isolated human thymocytes and in thymocytes activated with various inducing agents. In freshly isolated thymocytes c-myc mRNA is expressed at low levels, while thymocytes activated with Concanavalin A (Con A), the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), Con A in combination with TPA or interleukin 2 (IL-2) are induced to express higher levels of c-myc mRNA. The expression of c-myc is increased within 3 h of stimulation with these inducing agents; the amount of c-myc mRNA which is accumulated is not correlated with the rate of thymocyte proliferation. Dexamethasone and Cyclosporin A (CsA) which inhibit early events of T cell activation and the expression of the interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) genes also markedly suppress the expression of c-myc mRNA in Con A, and Con A + TPA-activated thymocytes. We conclude that activation of c-myc gene expression is an early event observed in activated human thymocytes. The level of c-myc expression is dependent on the mode of thymocyte activation rather than on the rate of thymocyte proliferation. Since freshly isolated thymocytes express low levels of c-myc mRNA it is possible that IL-2 induces c-myc expression at least in a responsive subpopulation of thymocytes during ontogeny.

Induction of interleukin 2 receptors on immature human thymocytes and co-expression of T3 and T6 antigens.

We have recently demonstrated that human thymocytes can be induced to express interleukin 2 (IL-2) receptors and to synthesize IL-2. The present study shows that relatively immature T6+ human thymocytes as well as the more mature T3+ thymocytes could be induced to express functional IL-2 receptors when activated with either Concanavalin A (Con A), Con A and 12-O-tetradecanoylphorbol 13-acetate (TPA) or IL-2 in combination with Con A or TPA. The phenotype of the common, immature thymocyte was identified by the binding of either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal anti-T6 (OKT 6) antibody and of mature thymocytes by the binding of monoclonal anti-T3 (OKT 3) antibody. We also observed that the expression of the T3 antigen on thymocytes, freshly isolated from thymic specimens obtained in the course of cardiac surgery of infants and children, was greater than 40% in 14 of 18 donors and that thymocytes co-expressed the T3 and the T6 antigen as determined by dual colour cytofluorometry. In thymocytes activated in vitro the expression of IL-2 receptors, determined by dual colour cytofluorometry with the PE-conjugated monoclonal anti-human IL-2 receptor antibody (PE anti-IL-2 R), was detected by the second day of induction in both immature T6+ and mature T3+ thymocytes. T6+ thymocytes proliferated in response to IL-2 and persisted in cultures for the duration of the study (18 days) and continued to express IL-2 receptors.

Induction and upregulation by interleukin 2 of high-affinity interleukin 2 receptors on thymocytes and T cells.

We show that purified recombinant interleukin 2 (rIL-2) alone induces the expression of high- and low-affinity interleukin 2 (IL-2) receptors in vitro on human T cells and thymocytes that have not been activated previously by lectins or other inducing agents. IL-2 receptors are expressed after 24 hr, as determined by the binding of 125I-labeled monoclonal anti-IL-2 receptor antibody 2A3, which binds equally to high- and low-affinity receptors. High-affinity receptors were distinguished from low-affinity receptors by the binding of 125I-labeled IL-2 to T cells and by the proliferative response of thymocytes to IL-2, in concentrations that selectively interact with the high-affinity class of IL-2 receptors. The IL-2-induced proliferation of thymocytes in vitro induced by IL-2 alone is dependent upon the concentration of IL-2 and is inhibited by monoclonal anti-Tac antibody, indicating that the proliferative response is mediated by the binding of IL-2 to the receptors. In addition, we demonstrate that IL-2 augments the number of high-affinity receptors on concanavalin A-activated thymocytes. These results document that IL-2 acts as a hormone that induces the activation of thymocytes and T cells, as evidenced by the de novo induction of biologically active, high-affinity IL-2 receptors. IL-2 also upregulates the expression of high-affinity IL-2 receptors on activated thymocytes. These observations illustrate the biologic importance of the regulatory role of IL-2 in the immune response.

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2.

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2.

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes.

Interleukin 2 (IL-2) has an important role in the regulation of the expression of IL-2 receptors and the synthesis of gamma interferon (IFN-gamma) by T lymphocytes. IL-2 is required for the optimum expression of IL-2 receptors on activated T lymphocytes and for maximum synthesis of IFN-gamma in vitro. Dexamethasone, an immunosuppressant drug that inhibits IL-2 synthesis, diminished the expression of IL-2 receptors and the synthesis of IFN-gamma. Anti-Tac, a monoclonal antibody known to prevent the binding of IL-2 to its receptor without inhibiting IL-2 synthesis, down-regulated the expression of the receptor and partially inhibited synthesis of IFN-gamma. In a population of T lymphocytes prevented from synthesizing IL-2 by dexamethasone and incapable of using IL-2 as a result of blockage of IL-2 receptors by anti-Tac, the number of receptor-bearing cells and receptor density were diminished. Anti-Tac in combination with dexamethasone also exerted a synergistic effect on IFN-gamma synthesis, inhibiting it almost completely. The inhibitory effect of dexamethasone IFN-gamma synthesis may be of clinical importance, since IFN-gamma activates macrophages and thereby triggers one of the defense mechanisms against bacterial infections.

Regulation of interleukin-2 synthesis in human thymocytes.

This study was designed to investigate whether or not human thymocytes can synthesize and respond to interleukin-2 (IL-2). Thymocytes were isolated from thymic sections obtained during cardiac surgery, and immature, heavy (density, 1.070-1.075 g/ml) thymocytes were separated from mature, light (density, less than or equal to 1.065 g/ml) thymocytes by buoyant density centrifugation. Thymocytes were stimulated with concanavalin A (con A), B lymphoblastoid (Bl) cells, phorbol myristate acetate (PMA), or with a combination of two or more inducing agents. Culture supernatants were analyzed for IL-2 activity by measuring the proliferation of an IL-2-dependent cytotoxic T cell line ( CTLL -A11). The response of human thymocytes to endogenous or exogenous (purified) IL-2 was assessed by determining their proliferative activity and their capacity to consume and absorb IL-2. Stimulation with con A in combination with PMA or Bl cells resulted in secretion of IL-2 by dense, immature thymocytes, low-density, mature thymocytes, and unfractionated thymocytes, and in an increase in [3H]thymidine incorporation. IL-2 synthesis preceded the proliferative response and required the continuous presence of con A. It was inhibited by cyclosporin A (CsA), an immunosuppressant which inhibits T cell activation and proliferation. As a result of the inhibition of endogenous IL-2 synthesis, thymocyte proliferation at 120 h of culture was also inhibited. CsA did not inhibit proliferation at 48 h of culture when exogenous IL-2 absorption became evident, but before the onset of the IL-2-dependent proliferative response.

Modulation of the expression of Tac antigen (T-cell growth factor response) on human thymocytes.

This study shows that anti-Tac antibody does not bind to human thymocytes unless they are activated. Human thymocytes could be induced to express Tac antigen (TCGF receptor) on their cell surface by Concanavalin A. B lymphoblastoid cells or 12-O-tetradecanoylphorbol 13-acetate alone did not induce TCGF receptors, but they did exert a very marked synergistic effect with Concanavalin A. This observation is consistent with our earlier finding that B lymphoblastoid cells secrete a factor which exerts a synergistic effect on the induction of lymphokine secretion by thymocytes and T-cells. The early expression of Tac antigen was independent of thymocyte proliferation. Anti-Tac antibody (10(-3)) inhibited the expression of TCGF receptors and late proliferation of thymocytes. TCGF did not reverse this inhibition nor did it prevent the binding of Tac antibody, but it enhanced the expression of Tac antigen.