Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes

The effect of phorbol esters on the proliferation and survival of interleukin-2(IL-2)-dependent cells was studied using an IL-2-dependent T cell line (CTLL-2) and blasts of BALB/c mouse spleen cells stimulated with Concanavalin A. Addition of phorbol 12,13-dibutyrate (PDBu) to CTLL-2 or ConA blasts induces a mitogenic response which is 25-40% of that elicited by IL-2. Interleukin 2 deprivation leads to a marked decline in the number of viable cells (50% of CTLL-2 cells have died after 8-10 hours incubation in IL-2-free medium). The mechanism of cell death seems to correspond to the suicide process known as apoptosis since an early degradation of DNA into oligonucleosome-size fragments could be observed after removal of the growth factor. When present, PDBu inhibits both the activation of the endonuclease and the development of the cell death process in CTLL-2 cells and ConA-blasts deprived of IL-2. Taken together, our results suggest that the tumor promoters phorbol esters inactivate in T cells the mechanism of cell elimination triggered by IL-2 deprivation and may help to explain why transformation of T cells decreases or even abolishes their requirements of IL-2 for survival and growth.     

Establishment of a cyclic adenosine monophosphate-dependent growing human T-cell line derived from an interleukin-2-dependent cell line

An adenosine 3',5'-cyclic monophosphate (cAMP)-dependent growing cell line called CT-Mat was established by the long-term cultivation of an interleukin-2 (IL-2)-dependent human T-cell line, ILT-Mat, in the presence of cholera toxin instead of IL-2. CT-Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT-Mat cell line can still grow dependent on IL-2, the forskolin-induced growth of CT-Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL-2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL-2. These suggest that cAMP transduces intracellular growth signals different from those through the IL-2 receptor in an IL-2-dependent T-cell line CT-Mat.     

Phorbol myristate acetate-induced proliferation of an IL-2-dependent T-cell line: action of PMA is independent of IL-2 and cannot be mimicked by diacylglycerols

Phorbol myristate acetate (PMA) at concentrations of 2 X 10(-10) to 2 X 10(-7) M was able to sustain proliferation of the interleukin 2 (IL-2)-dependent murine T-cell line CTLL-K. This line, which died within 24 hr without exogenously added IL-2, survived for at least 96 hr and completed two to three cycles of replication in the presence of an optimal dose of PMA. PMA did not increase proliferation induced by saturating amounts of IL-2, but it mimicked IL-2 activity when no IL-2 or only suboptimal doses of IL-2 were present. This effect was completely independent of any residual IL-2 activity and was not mediated by the endogenous production of IL-2. The finding that stimulation of CTLL-K cells with 1-oleoyl-2-acetyl-glycerol or 1,2-dioctanoyl-glycerol rather than with PMA was not sufficient to induce any proliferation suggests that diacylglycerols and phorbol esters have qualitatively different effects on protein kinase C activity. Falsely positive results could occur when IL-2-dependent T-cell lines were used as indicator cells for IL-2, since evidence is presented that PMA-responsive cells could emerge spontaneously from T-cell clones that originally were not responsive or only weakly influenced by PMA.     

Regulation of growth of an interleukin 2 (IL-2)-dependent murine T-cell clone (HT-2) in a defined serum-free medium

In this study, we demonstrate that an IL-2-dependent T-cell clone (HT-2) can be grown in a serum-free medium (HB101) with defined additives at rates comparable to those which can be obtained in serum-containing medium. Further, we show that cells cultured in the serum-free medium in the absence of IL-2 arrest growth in the G1 portion of the cell cycle, and that these arrested cells can be stimulated to reenter the cell cycle upon the addition of IL-2 to the culture medium. Growth of these cells in the absence of serum requires the presence of IL-2 as well as other hormones and growth factors and 2-mercaptoethanol. HT-2 cells have been grown continuously in the serum-free medium for periods of up to 1 month.     

Phosphorylation of human interleukin-2 (IL-2)

Human interleukin-2 (IL-2) is a lymphokine which is capable of activating lymphocytes and supporting the long-term in vitro growth of activated T cell clones. Recombinant human IL-2, expressed in either E. coli or cos cells, was shown to be phosphorylated by protein kinase C. Phosphorylated IL-2 synthesized in E. coli was analyzed by SDS-PAGE, reverse phase HPLC, and tryptic peptide mapping. The phosphorylated tryptic peptide was identified as the N-terminal fragment containing a single phosphorylation site at the serine residue at position 7. There was no difference in biological activity between non-phosphorylated and phosphorylated IL-2, as determined by a T cell growth assay. Although the physiological role of phosphorylation of IL-2 is unclear, IL-2 can be labeled with [-^32p] ATP and protein kinase C to a high specific radioactivity, and the synthesis of biologically active ^32p-labeled IL-2 may be useful for receptor-binding studies of the cells containing low level of phosphoprotein phosphotases.Abbreviations IL-2 Interleukin-2 - rIL-2 recombinant IL-2 - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis, Tris tris (hydroxymethyl)-amino methane - RP-HPLC Reverse Phase High Pressure Liquid Chromatography - PTH Phenylthiohydantoin - IFN- Leukocyte Interferon - IFN- Fibroblast Interferon - IFN- Immune Interferon By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleResearch being carried out at the Frederick Cancer Research Facility, Frederick, Maryland     

Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein.

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype.     

SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.     

Internalization of interleukin 2 (IL-2) by high affinity IL-2 receptors is required for the growth of IL-2-dependent T cell lines

During the growth of interleukin 2 (IL-2)-dependent T cells IL-2 binding is followed by internalization of the complex between IL-2 and the high affinity IL-2 receptor (HA-IL-2R). The respective role of IL-2 binding to HA-IL-2R and internalization of the complex has been examined. Monoclonal antibody 7D4 (IgM) blocks IL-2-dependent T cell growth although it does not affect IL-2 binding to HA-IL-2R. We show here that 7D4 inhibits T cell growth by blocking IL-2 internalization by HA-IL-2R. In contrast, Fab fragments prepared from 7D4 neither block IL-2 internalization nor inhibit T cell growth. Monoclonal 5A2, that recognizes an epitope related to the IL-2 binding site as well as its Fab fragment, inhibits T cell growth and IL-2 internalization. Monoclonal antibody 7D4, because of its pentameric structure, probably aggregates the IL-2R at the T cell surface and therefore prevents it internalization. The data presented in this paper suggest that simple occupancy of HA-IL-2R by IL-2 is not sufficient to transduce the T cell growth signal; this signal is transmitted only after internalization of the IL-2/HA-IL-2R complex.     

Inhibitors of IL-2 production and IL-2 receptor expression in human leukemic T-cell line, Jurkat

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, suppressed interleukin 2 (IL-2) production and IL-2 receptor (IL-2R) expression of the human leukemic T-cell line, Jurkat, induced by 12-O-tetradecanoyl-phorbol-13-acetate and phytohemagglutinin-P. This effect was significant at 5 microM H-7 without loss of cell viability. Such activity was not observed with N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), a potent inhibitor of cGMP- and cAMP-dependent kinases, and a weak inhibitor of Ca2+-phospholipid-dependent protein kinase (protein kinase C). These findings suggest that protein kinase C is more closely associated with IL-2 receptor expression and IL-2 production of T cells than cGMP- or cAMP-dependent kinases. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, suppressed both IL-2 production and IL-2R expression. Cycrosporin A (Cy A), a potent immunosuppressive drug, markedly inhibited IL-2 production of Jurkat cells whereas it did not affect the IL-2R expression. Thus, the mechanism of action of Cy A appears to differ from that of the protein kinase inhibitor, H-7, and the calmodulin inhibitor, W-7.     

Transforming growth factors beta slow down cell-cycle progression in a murine interleukin-2 dependent T-cell line

Transforming growth factors beta (TGF-beta) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF-beta have been attributed to the interference of these molecules with the interleukin-2 (IL-2)-driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF-beta on IL-2-induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF-beta 1 and 2) on the IL-2-driven proliferation of a murine IL-2-dependent T-lymphocyte line (CTLL). The results showed that pTGF-beta 1 and 2 decreased 3H-thymidine incorporation in CTLL cells in a dose-dependent fashion (maximum decrease of 75-85%). Combined-time kinetic analysis of the effects of pTGF-beta on 3H-thymidine incorporation, cell growth, and cell-cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF-beta 1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell-cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF-beta 1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF-beta 1-treated cells were still in S-G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL-2 receptor (IL-2R) nor the expression of the transferrin receptor (TfR) was affected by TGF-beta. After 72 h of culture in the presence of pTGF-beta 1, the expression of the IL-2R and TfR was decreased. The data suggest that in CTLL cells TGF-beta initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF-beta-mediated decrease of IL-2-induced 3H-thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL-2R and/or TfR.     

Glucocorticoid, interleukin-2, and prostaglandin interactions in a clonal human leukemic T-cell line.

We have studied the growth effects of conditioned media, interleukin-2 and PGE prostaglandin analogs on the glucocorticoid-sensitive human leukemic T-cell clone, CEM-C7. After 4 days, the glucocorticoid dexamethasone at approximately 10 nM kills 50% of CEM-C7 cells. To test the hypothesis that glucocorticoid-mediated lymphocytolysis was due to suppression of lymphokine expression only, we attempted to protect CEM-C7 cells from lysis by provision of lymphokine(s). Conditioned media from interleukin-2 secreting Jurkat T-cells as well as the glucocorticoid-insensitive, but receptor positive clone, CEM-C1, failed to prevent lymphocytolysis; exogenous interleukin-2 also did not provide protection. There were complex, biphasic interactions between dexamethasone and the synthetic PGEs, enisoprost and enisoprost free acid. Low doses of enisoprost alone (0.01 to 1 microgram/ml) stimulated growth, and in combinations completely reversed the growth inhibitory effects of 10 nM dexamethasone. Higher concentrations of enisoprost were inherently lethal and were additive to the steroid effect. Thus the glucocorticoid-induced lymphocytolysis in this human leukemic T-cell line may be modified biphasically by PGE prostaglandins, depending on their concentration. However, interleukin-2 or components in the conditioned media assayed had no effect in ameliorating the lethal response to glucocorticoid.     

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits phorbol ester-induced growth of an IL-2-dependent T cell line

We previously established a human T cell line, TPA-Mat, which can proliferate in response to not only interleukin-2 (IL-2), but also phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). The present study demonstrated that the PDBu-dependent growth of TPA-Mat cells was inhibited up to 90% by adenosine 3',5'-cyclic monophosphate (cAMP] raising agents such as forskolin, cholera toxin and 1-methyl-3-isobutyl-xanthine, and cAMP analogues, whereas the IL-2-stimulated TPA-Mat growth was slightly inhibited. These findings suggest that the signal transduction pathway of PDBu-induced growth, which should involve activation of protein kinase C, is sensitive to cAMP, and that it cannot be exactly identical to the signal transduction pathway of Il-2-induced growth in TPA-Mat cells.     

Synergistic and antagonistic effects of IL-1 alpha and IL-4, respectively, on the IL-2-dependent growth of a T cell receptor-gamma delta+ human T leukemia cell line

The TALL-103/2 cell line was derived from an immature acute T lymphocytic leukemia with T-myeloid differentiating capacity. The leukemic cells were first expanded in recombinant human IL-3 in which they acquired a myeloid phenotype, and subsequently were adapted to grow in human rIL-2 in which they became lymphoid committed. The TALL-103/2 cell line expresses only T cell-specific differentiation Ag (CD2, CD3, CD7, and CD8) but has retained the CD33 myeloid Ag originally present on the IL-3 expanded population. By using mAb directed at the TCR-alpha beta or specific for framework determinants on human TCR-gamma and -delta chains, the TALL-103/2 cells were shown to be WT31-, TCR delta 1+, TCS-1+, and Ti gamma A-, thus representing a T cell subset expressing the nondisulfide-linked form of the TCR-gamma delta. The TALL-103/2 cells have been maintained for more than 1 y in the presence of human rIL-2 on which they are strictly dependent. Chemical cross-linking and immunofluorescence studies indicate the presence of both high and intermediate affinity IL-2R on the TALL-103/2 cells. Whereas mAb antiTac and H-31 with reactivity to the IL-2R alpha-chain (p55) compete only partially for the IL-2-induced proliferation of these cells, mAb TU27, specific to the IL-2R beta-subunit (p75), inhibits such growth completely even at high concentrations of IL-2. The interactions of the two T cell-stimulating factors IL-1 and IL-4 on the IL-2-dependent growth of TALL-103/2 cells were investigated. IL-1 alpha synergizes with IL-2 in supporting the short and long term growth of this cell line, whereas IL-4 abrogates its growth. These effects are, at least in part, due to the modulation of IL-2R expression induced by the two lymphokines. Functionally, the TALL-103/2 cells display MHC-nonrestricted cytotoxic activity that is significantly enhanced by addition of either IL-4, IL-6, or IFN-gamma. Because of its properties and its stable requirement for IL-2 for continuous growth, this T lymphocytic leukemia-derived cell line represents an interesting model to analyze ontogeny and function of leukemic T cells.     

Identification of structural differences between different forms of interleukin-2 (IL-2) using anti-(human recombinant) IL-2 monoclonal antibodies.

We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (Kaff approximately equal to 10(-8) M) and are of the IgG1 isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh IL-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IL-2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID50, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 micrograms/ml to 30 micrograms/ml final concentrations of the antibodies. Using different forms of IL-2 we found that the mAbs give different patterns of inhibition of the IL-2-dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2.     

Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.