Phorbol esters inactivate the lytic apparatus of cytotoxic T lymphocytes

Treatment of cloned murine CTL with phorbol myristate acetate (PMA) initiates the loss of lytic potential of these cells. The process is relatively rapid (t 1/2 = 2.2 +/- 0.7 hr), occurs independently of protein synthesis, and can be largely reversed within 3 hr by incubation in normal medium with or without the addition of supernatants from activated lymphocytes. Examination of PMA-treated or untreated CTL in assays that separate binding and post-binding events suggests that the lytic apparatus itself is a major target of the PMA-initiated event.     

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.     

IL-4 potentiates activated T cell apoptosis via an IL-2-dependent mechanism

Activation-induced cell death (AICD) of T cells is one of the major mechanisms of peripheral tolerance. The regulation of AICD by IL-4 is poorly understood. In this study, we report that AICD in IL-4-deficient T cells is significantly reduced compared with that in wild-type T cells. This impaired AICD correlates with the failure to induce degradation of cellular FLIP. IL-4-mediated enhancement of AICD and cellular FLIP degradation requires a Janus kinase/STAT-6 signaling pathway. Unexpectedly, these effects of IL-4 could be blocked by a neutralizing anti-IL-2 Ab, and addition of rIL-2 could completely restore the defective AICD in IL-4-deficient T cells. Furthermore, IL-4 regulates the T cell thresholds for IL-2 signaling during AICD. These data suggest that IL-4 promotes AICD via an IL-2-dependent mechanism.     

Glucocorticoids inhibit serum depletion-induced apoptosis in T lymphocytes expressing Bcl-2.

Depletion of growth factors and glucocorticoids are known to induce apoptosis and inhibit growth in T lymphocytes. We have examined the effect of Bcl-2 expression on the cellular response to growth factor depletion in the presence or absence of glucocorticoids. Cell growth was determined by cell counting and viability was quantitated by dye exclusion. Apoptosis was evaluated by flow cytometry, analysis of DNA integrity, and enzymatic determination of caspase-3-like activity. Serum depletion and glucocorticoid administration inhibited cell growth and stimulated apoptosis in Bcl-2 negative cells. Cotreatment with both stimuli had additive effects on apoptosis but not on inhibition of cell growth. Bcl-2 expression abrogated the repressive effect of glucocorticoids on apoptosis but not on cell growth. In contrast, neither apoptosis nor growth inhibition induced by serum depletion of cells was blocked by Bcl-2 expression. However, glucocorticoid treatment of Bcl-2-overexpressing cells protected them from apoptosis induced by serum depletion. Glucocorticoid protection of Bcl-2-overexpressing cells from serum depletion-induced apoptosis was mimicked by other inducers of apoptosis, which act to inhibit protein synthesis. These data suggest that Bcl-2 expression can switch the effect of glucocorticoids from proapoptotic to antiapoptotic when lymphocytes expressing Bcl-2 are exposed to other apoptotic stimuli.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Phorbol esters inhibit LH stimulated steroidogenesis by mouse Leydig cells in vitro

The effect of phorbol esters on the stimulation of testosterone production in response to LH was studied in mouse Leydig cells incubated in vitro. The tumor promoting phorbol esters, Phorbol-12-myristate-13-acetate and Phorbol-12-13-didecanoate at nanomolar concentrations effectively inhibited testosterone production by Leydig cells in response to stimulation by LH, whereas non-tumor promoting phorbol esters were ineffective. When the cells were stimulated by 8Br-cAMP, instead of LH, the testosterone production was stimulated similarly as in the presence of LH, but phorbol esters were without any effect. This suggests that the tumor promoting phorbol esters may act in the Leydig cells by suppressing the stimulation of cAMP production in response to hormonal activation and/or by interfering with the hormone-receptor interaction.     

Phorbol esters inhibit the synthesis of acetylcholine receptors in cultured muscle cells

The acetylcholine receptor (AChR) synthesis, insertion and degradation rates are regulated by numerous intracellular and extracellular agents. Recent studies have shown that Ca2+ and Ca2+ ionophores have a profound regulatory effect on the appearance of AChR clusters and AChR synthesis. These regulatory effects may be mediated through the activation of calcium and phospholipid-dependent protein kinases by agents such as phorbol esters. In this study, we have utilized 4-beta-phorbol-12-myristate-13-acetate (PMA) in order to determine whether the activation of protein kinase C exerts a regulatory effect on the expression of AChRs in cultured chick myotubes. Our results show that 4-beta-phorbol-12-myristate-13-acetate decreased intracellular AChRs and suppressed AChR synthesis without affecting the turnover rate. Control and PMA treated cells labeled with [35S] methionine and immunoprecipitated with a monoclonal antibody to the alpha subunit of AChRs (mAb35) revealed a significant decrease in radioactivity precipitated after exposure to PMA. Polyacrylamide gel electrophoresis revealed no major changes in protein patterns, or in newly synthesized proteins as determined by [35S] methionine incorporation and autoradiography. Other enzymes important in muscle metabolism were not affected by PMA treatment. Our results indicate that activation of protein kinase C results in the suppression of AChRs synthesis and dispersal of AChR clusters.     

Phorbol esters inhibit low density lipoprotein processing by cultured human fibroblasts

A 24 h pretreatment of MRC5 fibroblasts with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a marked decrease in low density lipoprotein (LDL) internalization and degradation; the maximal effect (about 55% decrease) was observed for 10(-7) M TPA. LDL binding was reduced about 35-40%. A significant decrease (about 25%) in LDL internalization was observed after a 2 h incubation of cells with the drug, but longer incubation times (4-6 h) led to a greater effect. Another tumor promoter, phorbol 12,13-dibutyrate decreased LDL internalization by about 35%, whereas the non-tumor promoting 4 alpha-phorbol 12,13-didecanoate had no effect. The protein kinase C inhibitor alpha-cobrotoxin partially antagonized the inhibitory effect of TPA on LDL internalization. The non-phorbol tumor promoter mezerein, another protein kinase C activator, decreased LDL uptake by about 50%. Finally, it was found that TPA had no significant effect on the affinity of the receptor for the LDL. These results suggest a role for protein kinase C in the LDL pathway in cultured human fibroblasts.     

Phorbol esters enhance spontaneous cytotoxicity of human lymphocytes, abrogate Fc receptor expression, and inhibit antibody-dependent lymphocyte-mediated cytotoxicity

Phorbol esters with tumor promoter activity enhance the spontaneous cytotoxicity of human lymphocytes against a variety of target cell lines, with an efficiency that correlates with their potency as tumor promoters or skin irritants. Analysis of surface marker expression of the lymphocytes cytotoxic after treatment with phorbol ester identified the cytotoxic cell subset as that containing natural killer cells. Although gamma-interferon (IFN gamma) is produced by T cells treated with phorbol esters, IFN gamma is probably not the mediator of enhancement of natural killer cell activity, because anti-IFN gamma antibodies failed to block this enhancement. Spontaneous cell-mediated cytotoxicity is inhibited when phorbol esters are present during the cytotoxic assay, but is enhanced when the effector cells are pretreated with these agents. On the other hand, antibody-dependent cytotoxicity mediated by lymphocytes is inhibited by phorbol ester pretreatment of the effector cells or by phorbol esters present during the cytotoxic assay. Treatment of lymphocytes with phorbol esters at 37 degrees C, but not at 4 degrees C, completely abrogates in 1 to 2 hr the expression of the receptor for the Fc fragment of IgG, as detected by rosette formation with IgG-sensitized erythrocytes and by reactivity with anti-Fc receptor antibodies. The inhibition of antibody-dependent cytotoxicity by phorbol esters is probably secondary to their effect on the Fc receptor.     

Phorbol esters and diacylglycerol inhibit vasopressin-induced increases in cytoplasmic-free Ca and Ca efflux in Swiss 3T3 cells

Vasopressin increased intracellular free calcium concentration [Ca²⁺]_i in quin-2-loaded quiescent Swiss 3T3 cells. This effect of vasopressin was rapidly inhibited by biologically active tumour promoters including phorbol dibutyrate (PBt₂) and by the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG). Prolonged pretreatment of Swiss 3T3 cells with PBt₂ causes a loss of protein kinase C activity (Rodriguez-Pena & Rozengurt, Biochem biophys res commun 120 (1984) 1053) [28]. This pretreatment abolished the inhibition by PBt₂ or OAG of vasopressin-mediated increases in Ca²⁺]_i. Vasopressin also stimulated ⁴⁵Ca²⁺ efflux from cells pre-loaded with the isotope. This effect of the hormone was also inhibited by PBt₂. Prolonged pretreatment with PBt₂ prevented the inhibition of vasopressin-stimulated ⁴⁵Ca²⁺ release by PBt₂. Thus, protein kinase C stimulation inhibits vasopressin-mediated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ efflux apparently by blocking the increased release of Ca²⁺ from an intracellular store caused by the hormone. These findings suggest that activation of protein kinase C may act as a feedback inhibitor to modulate ligand-mediated increases in [Ca²⁺]_i.     

Phorbol esters inhibit the functional activation of cytotoxic precursors in mixed lymphocyte cultures

We have investigated the effect of phorbol esters on T cell activation and generation of suppressor and cytotoxic activity in mixed lymphocyte cultures (MLC). The presence of 30 nM P(Bu)2 during the sensitization phase inhibited the generation of allospecific cytotoxicity and also decreased the killing potential against NK-sensitive targets. The inhibition was not mediated by direct blocking of the lytic capacity nor by suppression of clonal expansion of cytotoxic cells through modulation of lymphokine production. The presence of P(Bu)2 enhanced cell proliferation, but inhibited the functional activation of lymphocytes and consequent generation of Dr antigen-positive T cells. Because the presence of the compound did not affect the MLC-induced generation of suppressor activity, it is likely that P(Bu)2 selectively blocks the maturation of cytotoxic precursors. Surface-marker analysis with OKT monoclonal antibodies revealed that the effects on lymphocyte activation were associated with a decrease in OKT3 and OKT4 reactivity and an increase in the percentage of OKT8-positive cells. The decrease in OKT4 reactivity was not due to selective loss of this lymphocyte subpopulation, because P(Bu)2 was equally mitogenic for the purified OKT4- and OKT8-positive subsets. The results suggest that the effect of P(Bu)2 on cell differentiation and its ability to modulate the expression of functional markers in lymphocyte subsets may interfere with T-T cell cooperation that controls the functional maturation of cytotoxic precursors.     

IL-21 induces apoptosis of antigen-specific CD8+ T lymphocytes

IL-21, a member of the common gamma-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common gamma-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.     

Agents that mimic antigen receptor signaling inhibit proliferation of cloned murine T lymphocytes induced by IL-2

We have shown previously that stimulation of cloned murine T lymphocytes via the TCR inhibits their responsiveness to rIL-2. Signaling via the TCR is believed to result in a variety of biochemical events that include a rise in intracellular free calcium and activation (translocation) of protein kinase C. These two signals also can be generated by calcium ionophores, such as ionomycin, and by activators of protein kinase C, such as PMA. We report here that treatment of cloned murine T lymphocytes with PMA, ionomycin, or the combination led to a dose-dependent inhibition of IL-2-dependent proliferation but did not inhibit lymphokine secretion. Concentrations of PMA and ionomycin that maximally inhibited proliferation stimulated maximal lymphokine secretion and increased mitochondrial activity as assessed by measurement of cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide. Furthermore, PMA, ionomycin, the combination, or immobilized anti-CD3 mAb added after 12 to 16 h of culture with IL-2 could inhibit proliferation. These results demonstrate that PMA and ionomycin mimic stimulation of the TCR by high concentrations of immobilized anti-TCR mAb in that proliferation is inhibited and lymphokine secretion is induced. In addition, PMA or ionomycin could independently inhibit proliferation of some cells. These findings suggest that alternative mechanisms exist to regulate proliferation. Either increased levels of intracellular calcium or the physiologic events corresponding to those induced by PMA can inhibit IL-2-dependent replication of T lymphocytes.     

IL-10-dependent suppression of experimental allergic encephalomyelitis by Th2-differentiated, anti-TCR redirected T lymphocytes

We previously showed that transgenically expressed chimeric Ag-MHC-zeta receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic zeta-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IAs-zeta receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8+ RMTC were therapeutically effective whereas CD4+ RMTC were not. We re-examine here the therapeutic potential of CD4+ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4+ MBP89-101-IAs-zeta RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.     

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP1

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E2 (PGE2) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE2 and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE2 and cAMP levels increased progressively during the 6 days of days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in chondrogenesis which occur independent of those initiated by PGE2 and the cAMP system.     

IL-2 dependence of IL-9 expression in human T lymphocytes.

Human IL-9 is a T cell-derived lymphokine that is abundantly expressed upon activation with mitogens. The observation that IL-9 induction peaks as late as 28 h after stimulation suggested the involvement of secondary signals in this process. The finding reported here that IL-9 expression is blocked by cycloheximide strongly supports this hypothesis. Moreover, we identify IL-2 as the critical element controlling IL-9 expression in T cells. We show (i) that anti-IL-2R antibodies block IL-9 expression in T cells stimulated with PMA and anti-CD3 and (ii) that IL-2, of a panel of cytokines, is the only molecule that synergizes with PMA for IL-9 induction. The latter finding is confirmed in a T cell leukemia line. Finally, we demonstrate that IL-2 plays a regulatory role in the induction of other cytokines, such as IL-4, IL-5, IL-6, and granulocyte/macrophage-CSF, in fresh peripheral T cells.