Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes

Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells.     

Response of Jurkat T cells to phorbol ester and bryostatin. Development of sublines with distinct functional responses and changes in protein kinase C activity.

T lymphocyte activation is initiated as a result of the interaction between the TCR complex and Ag as seen in the framework of a membrane-bound MHC molecule. Receptor stimulation results in a rise in free intracellular Ca2+ and the activation of protein kinase C (PKC). Bryostatin (Bryo) and phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA] are PKC activators with somewhat different immunologic effects. We compared the effect of Bryo and TPA on the T cell tumor line Jurkat and derivatives of Jurkat cells grown in media supplemented with 100 nM Bryo ("BR100" cells) or 100 nM TPA ("TP100" cells). In untreated Jurkat cells, there is a dose- and time-dependent decrease in proliferation, compared to media controls, after the administration of as little as 10 nM TPA. This can be reversed in a dose- and time-dependent manner by Bryo. Interestingly, the expression of the transferrin receptor parallelled this effect on proliferation. Furthermore, Jurkat cells grown continuously in 100 nM TPA regained full proliferative capacity after several weeks in culture and transferrin receptor expression returned to near the level seen in untreated Jurkat cells. The chromatographic separation of PKC activity in these three cell lines showed that total PKC activity was dramatically decreased in both the TP100 and BR100 cells when compared to untreated Jurkat cells. However, in the TP100 cells there exists a peak of activity that is activated by Bryo, but not TPA. Western blots of whole cell lysates of the three cell lines showed that PKC-alpha and PKC-beta II were both down-regulated in BR100 and TP100 cells compared to untreated Jurkat cells. PKC-gamma was not detected in any of the cell lines. Therefore, the Bryo-specific peak seen in TP100 cells may be PKC-delta, -epsilon, -zeta, -eta, or a novel PKC isoform. This could provide the basis for a molecular characterization of the differences in PKC activation between phorbol esters and Bryo.     

The role of protein kinase C activation in signal transmission by interleukin 2

Role of protein kinase C (PKC) in interleukin (IL) 2-induced proliferation was investigated by utilizing two murine IL 2-dependent cell lines, CT6 and CTLL-2 cell lines. CT6 cells showed a marked proliferative response to phorbol 12-myristate 13-acetate (PMA), while CTLL-2 did not. PMA induced PKC translocation from cytosol to membrane only in a PMA-responsive cell line. IL 2 failed to stimulate PKC translocation in both cell lines. H-7, a potent and specific PKC inhibitor, however, inhibited the proliferation of both cell lines induced by IL 2. Taken collectively, IL 2 may induce PKC activation without its translocation.     

Association of protein kinase C activation with IL 2 receptor expression

Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK-C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression.     

Synergistic action of phorbol ester and IL-3 in the induction of "connective tissue-type" mast cell proliferation

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induced the proliferation of connective tissue-type mast cells (CTMC) synergistically with IL-3 in a methylcellulose culture, as well as with IL-4. The culture of single CTMC and the serum-free culture of CTMC fractionated by Percoll density gradient centrifugation showed that this synergistic action of IL-3 and TPA required no effects of accessory cells or other humoral factors. Although the populations of CTMC acted on by TPA and IL-4 seemed to be close to each other, the velocity of colony growth induced by the simultaneous stimulation of the combination of TPA and IL-4 was faster than that induced by either TPA or IL-4 in the presence of IL-3. In addition, the addition of anti-IL-4 antibody did not neutralize the effect of TPA on the proliferation of CTMC. These results suggest that TPA and IL-4 act on the proliferation of CTMC synergistically with IL-3 via a different pathway. Beside TPA, other phorbol derivatives capable of activating protein kinase C (PKC) induced the proliferation of CTMC synergistically with IL-3, but phorbol derivatives which were unable to activate PKC did not. These results indicate that the activation of PKC is involved in the process of TPA action on the proliferation of CTMC. Furthermore, the facts that 1-oleoyl-2-acetylglycerol, which activated membrane PKC transiently, and staurosporine, which has been reported to inhibit PKC, did not induce the proliferation of CTMC in the presence of IL-3 and that the effect of TPA was exhibited by the sustained stimulation suggest that the action of TPA on the proliferation of CTMC requires at least two steps. The first one is the primary activation of membrane PKC and the second one is the disappearance of PKC from the cells, "down-regulation."     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

An intracellular calcium increase and protein kinase C activation fail to initiate T cell proliferation in the absence of a costimulatory signal

Resting T lymphocytes proliferate in response to a combination of a calcium ionophore and a phorbol ester. This observation suggests that an increase in intracellular calcium free ion concentration [Ca2+]i and activation of protein kinase C (PKC) are sufficient signaling events for the initiation of T cell proliferation. In contrast, an accessory cell-generated costimulatory signal, acting independently of the rise in [Ca2+]i and PKC activation, is required for Ag-induced proliferation of type I T cell clones. We now report that this costimulatory signal is unexpectedly also being delivered via a cell-cell interaction during the response to ionomycin and phorbol ester. In the absence of this signal (at limiting cell numbers), T cells fail to divide. We also demonstrate that proliferation in response to immobilized anti-CD3 mAb requires the cell-cell interaction. These results suggest a model of T cell stimulation in which activation of a costimulatory signaling pathway is important in the regulation of the IL-2 gene, and only in the presence of this (third) signal can an increase in [Ca2+]i and PKC activity induce T cell proliferation. Such a model predicts that IL-2-dependent expansion of T cell clones in vivo in response to Ag receptor occupancy requires the delivery of an independent accessory cell-derived co-stimulatory signal.     

Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors

Previous studies indicated that Ca++ ionophores and phorbol esters in synergy could substitute for the initial activation step of normal T lymphocytes or T cell clones leading to increased expression of receptors for the growth factor interleukin 2 (IL 2) and secretion of interleukins, with the mitogenic signal for T cell proliferation being dependent on the presence of IL 2. In this study, the question was addressed as to whether T lymphocytes activated through the Ca++ ionophore ionomycin and the phorbol ester 12-o-tetradecanoyl phorbol 3-acetate (TPA) also acquired the competence to kill relevant target cells. The results indicate that T lymphocytes from primed mice proliferate and lyse the relevant allogeneic target cells after in vitro stimulation with ionomycin plus TPA, and that T lymphocyte preparations enriched for a subpopulation bearing the Lyt-2 marker are dependent on exogeneous sources of IL 2 to proliferate and become competent killer cells, whereas preparations enriched for subpopulations bearing the L3T4 marker grow independently of exogenous IL 2.     

Translocation of protein kinase C is not required to inhibit the antigen-induced increase of cytosolic calcium in a mast cell line

Cross-linking of receptor bound IgE antibodies by multivalent antigen (DNP8-BSA) on PB-3c cells leads to an increase of cytosolic calcium ((Ca2+)i). Active tumor promoting phorbol esters and teleocidin which specifically activate the phospholipid Ca2+-sensitive protein kinase (PKC), inhibited the antigen-mediated rise in (Ca2+)i and induced a time and dose-dependent translocation of cytosolic PKC to membranes of the PB-3c cells as determined by enzyme activity or immunoblotting using a polyclonal anti-PKC antibody. This TPA concentration did not affect the subcellular distribution of PKC, although 1 nM of 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited to 50% the antigen-mediated increase in (Ca2+)i. The concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the antigen-mediated increase in (Ca2+)i. These data demonstrate that the TPA-dependent activation of PKC is not directly coupled to its translocation to membranes.     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.     

TPA-induced signal transduction: a link between PKC and EGFR signaling modulates the assembly of intercellular junctions in Caco-2 cells

Recent studies suggest that signal transduction may have an important role in the development and regulation of the metastatic phenotype. Here, we investigated the role of the epidermal growth factor receptor (EGFR), and protein kinase C (PKC), in the process of reassembly of cadherin-dependent cell-cell adhesion of Caco-2 cells. We used chemical activation of PKC and EGFR with 12- O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting agent, pretreatment with protein kinase inhibitors and subcellular fractionation to analyze the effect of the phorbol ester on the redistribution of junctional proteins. Transepithelial resistance (TER), electron microscopy and immunofluorescence analyses were also carried out. Activation with TPA resulted in disassembly of adherens junctions (AJs), but the tight junction (TJ) structure and function remained unaltered. TPA affected E-cadherin levels. In Caco-2 cells at day 2 of culture, when most E-cadherin is not associated with the cytoskeleton, a decrease in the level of this protein was observed as soon as 6 h after TPA addition. However, at day 5 of culture, the major effect observed after 6 h of treatment was a translocation of the protein from the Triton-insoluble to the -soluble fraction. On the other hand, TPA did not significantly affect the E-cadherin-associated proteins alpha and beta-catenins. Potent specific EGFR inhibitors, such as PD153035 and Tyrphostin 25, as well as Calphostin C, an inhibitor of PKC, significantly blocked the effect of TPA on AJs. Furthermore, inhibition of the TPA effect by the PD98059 MAPK inhibitor suggests that activation of this kinase was the final event in the modulation of cadherin-dependent cell-cell adhesion. Pretreatment of cell monolayers with Calphostin C before EGF treatment, one of the ligands of EGFR, blocked the redistribution of E-cadherin caused by EGF. Based on these results, we conclude that both EGFR and PKC activation are involved in TPA-induced cell signaling for modulation of cadherin-dependent cell-cell adhesion and cell shape in Caco-2 cells.     

Teleocidin and phorbol ester tumor promoters exert similar mitogenic effects on human lymphocytes

The tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) affects a wide variety of cellular functions via its binding to protein kinase C (PKC). The TPA molecule contains a diacylglycerol (DAG)-like structure, which may explain its ability to mimic DAG in PKC activation. Teleocidin (TCD) is a different tumor promoter which can compete with TPA in binding to its cell surface receptors even though structurally unrelated to TPA or DAG. Since TCD may use an additional receptor system and/or be distinguished from TPA in its effect on cells, we compared the effects of TPA and TCD on human peripheral blood lymphocytes (PBL). Both tumor promoters preferentially enhanced cell proliferation of sheep erythrocyte-rosetted lymphocytes, which were enriched for T cells. Additionally, TPA and TCD both induced a high density of cell surface receptors for interleukin 2 (IL2) and transferrin, but not synthesis or production of IL2. However, either of the tumor promoters synergized with T cell mitogens to induce high level IL2 production by PBL. In dose response and kinetic studies, matching concentrations of TPA and TCD induced similar effects in PBL. The results thus demonstrate that TPA and TCD are alike in mitogenic capacity, and suggest that structural similarity between the tumor promoter and DAG, the physiological activator of PKC, is not an essential property for promoting tumors or affecting a wide variety of cellular functions.     

Phorbol esters can persistently replace interleukin-2 (IL-2) for the growth of a human IL-2-dependent T-cell line

A selected clone from an IL-2-dependent human T-cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutylate (PDBu). Thus, a TPA(PDBu)-dependent T-cell line, designated TPA-Mat, was established from IL-2-dependent T cells. The TPA-dependency of TPA-Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA-Mat cells still showed IL-2-dependent growth. However, the TPA (PDBu)-dependent growth of TPA-Mat did not seem to be mediated by an autocrine mechanism of IL-2 or by any other growth factor production, because these factors were not detected in TPA-Mat cell supernatants. Therefore, the phorbol esters substituted for IL-2 and may be directly involved in transduction of growth signals in TPA-Mat cells. Although activity of PKC was down-regulated, messenger ribonucleic acid (mRNA) of the PKC beta-gene was detected in TPA-Mat cells cultured with PDBu. Furthermore, the growth of TPA-Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL-2-dependent TPA-Mat cells.