Coordination and reversibility of signals for proliferative activation and interleukin-2 mRNA production in resting human T lymphocytes by phorbol ester and calcium ionophore (original) (raw)
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Journal of Clinical Immunology, 1994
To determine a role of protein kinase C (PKC) isozymes in lymphocyte activation, human peripheral blood mononuclear cells were activated with 12Meoxyphorbol-13-Ophenylacetate (dPP, an agonist of both calciumdependent and calcium-independent PKC isozymes), thymeleatoxin (TX; an activator of calcium-dependent PKCax, 13, and ~/), and 12-deoxyphorbol-13-O-phenylacetate 20 acetate (dPPA; an activator of PKC131 isozyme) and examined for DNA synthesis, lymphocyte proliferation, interleukin-2 (IL-2) production, expression of IL-2 receptor ot and 13 chains on CD3+, CD4+, and CD8+ T lymphocytes and CD20+ B lymphocytes, and translocation of PKC13 isozyme from cytosol to membrane fraction. The results show that dPPA activates lymphocytes by inducing the above changes in a manner analogous to that of dPP, TX, and phorbol myristate acetate. These data suggest that PKC131 is involved in the activation of human peripheral blood T and B lymphocytes. . 3Abbreviations used: dPP, 12-deoxyphorbol-13-O-phenylacetate; dPPA, 12-deoxyphorbol-13-O-phenylacetate-20-acetate; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; HBSS, Hanks' balanced salt solution; IL-2, interleuldn-2; IL-2R, interleukin-2 receptor; MNC, mononuclea r ceils;MTT, 3-(4,5-dimethythiazol-2yl)2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PE, phycoeiythrin; PKC, protein kinase C; PMA, phorboi myristate acetate; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoyl phorbol-13-acetate; TX, thymeleatoxin.
Cellular Immunology, 1988
The proliferative responses of natural killer (NK) cells to 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly activates protein kinase c (PKC), and to the Ca*+ ionophores A238 17 and ionomycin, known to enhance the intracellular calcium, have been investigated. Highly purified large granular lymphocytes (LGL) were cultured for 12-30 hr in the presence of TPA, ionomycin, or A238 17. TPA alone (I-20 rig/ml) triggered rapid LGL proliferation, whereas the calcium ionophores were ineffective. The addition of either calcium ionophore to suboptimal doses or TPA (0.1-0.5 @ml) resulted in a synergistic effect on LGL proliferation. Under these conditions high levels of IL-2 activity were released by the LGL. Phenotypic analysis revealed the rapid loss of the Fey receptors (CD 16) on LGL and the induction of the expression of IL-2 (CD25) and transfenin receptors and of HLA-DR, but not of CD3. Removal of extracellular Ca*+ by addition of EGTA at the beginning of the culture greatly depressed LGL proliferation and IL-2 production, and blocked phenotypic changes, such as the expression of Tat antigen. Finally, progression to the proliferative phase of LGL, activated by TPA alone or with ionomycin, was completely abrogated by a hyperimmune anti-IL-2 antiserum. o 1988 Academic Press, Inc.
Agents and Actions, 1992
The effects of the phorbol ester, phorbol-12-myristate-13-acetate (PMA), and of the calcium ionophore A23187 on DNA synthesis in murine quiescent and mitogen-stimulated lymphocytes have been examined. Neither PMA nor A23187 had any mitogen effect on their own on quiescent lymphocytes. However, stimulation of cells sequentially with A23187 and then PMA resulted in a proliferative response in proportion to the duration of the exposure to A23187, and the sustained simultaneous presence of both agents was necessary for maximum proliferation. On the other hand, while short incubations with A23187 potentiated mitogen-induced DNA synthesis, prolonged exposure inhibited it. Furthermore, on lymphocytes stimulated with two T cell mitogens, the effects of A23187 and PMA depended on the proliferation-inducing mitogens and the responsiveness level induced by them. Therefore, while PMA and short pretreatments with A23187 had no effect on high intensity mitogenic responses, low intensity responses were significantly enhanced. These results demonstrate differential effects of A23187 and PMA on DNA synthesis that should be useful in studies on the mechanisms of activation of lymphocyte proliferation.
Journal of Clinical Immunology, 1994
To determine a role of protein kinase C (PKC) isozymes in lymphocyte activation, human peripheral blood mononuclear cells were activated with 12-deoxyphorbol-13-O-phenylacetate (dPP; an agonist of both calcium-dependent and calcium-independent PKC isozymes), thymeleatoxin (TX; an activator of calcium-dependent PKCα, β, and γ), and 12-deoxyphorbol-13-O-phenylacetate 20 acetate (dPPA; an activator of PKCβ1 isozyme) and examined for DNA synthesis, lymphocyte proliferation, interleukin-2 (IL-2) production, expression of IL-2 receptor α and β chains on CD3+, CD4+, and CD8+ T lymphocytes and CD20+ B lymphocytes, and translocation of PKCβ isozyme from cytosol to membrane fraction. The results show that dPPA activates lymphocytes by inducing the above changes in a manner analogous to that of dPP, TX, and phorbol myristate acetate. These data suggest that PKCβ1 is involved in the activation of human peripheral blood T and B lymphocytes.
Immunology, 2009
The costimulatory receptors CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 and their ligands, CD80 and CD86, are expressed on T lymphocytes; however, their functional roles during T cell–T cell interactions are not well known. The consequences of blocking CTLA-4–CD80/CD86 interactions on purified mouse CD4+ T cells were studied in the context of the strength of signal (SOS). CD4+ T cells were activated with phorbol 12-myristate 13-acetate (PMA) and different concentrations of a Ca2+ ionophore, Ionomycin (I), or a sarcoplasmic Ca2+ ATPase inhibitor, Thapsigargin (TG). Increasing concentrations of I or TG increased the amount of interleukin (IL)-2, reflecting the conversion of a low to a high SOS. During activation with PMA and low amounts of I, intracellular concentrations of calcium ([Ca2+]i) were greatly reduced upon CTLA-4–CD80/CD86 blockade. Further experiments demonstrated that CTLA-4–CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of I or TG (high SOS) but the opposite occurred with PMA and low amounts of I or TG (low SOS). These results were confirmed by surface T-cell receptor (TCR)–CD3 signalling using a low SOS, for example soluble anti-CD3, or a high SOS, for example plate-bound anti-CD3. Also, CTLA-4–CD80/CD86 interactions enhanced the generation of reactive oxygen species (ROS). Studies with catalase revealed that H2O2 was required for IL-2 production and cell cycle progression during activation with a low SOS. However, the high amounts of ROS produced during activation with a high SOS reduced cell cycle progression. Taken together, these results indicate that [Ca2+]i and ROS play important roles in the modulation of T-cell responses by CTLA-4–CD80/CD86 interactions.
Biochemical and Biophysical Research Communications, 1990
complex induces a rise in the intracellular concentration of Ca2+ within seconds. The inositol phosphate-dependent Ca2+ mobilization induced by OKT3 was completely abrogated when Jurkat cells were pretreated for 1 min with the phorbol 12-myristate 13-acetate TPA (10nM), a concentration which activates protein kinase C (PKC). The effects of TPA on the Ca2+ fluxes were insensitive to treatment of the cells with known PKC inhibitors ( H-7 and staurosporin) under conditions where the PKC-mediated phosphorylation was blocked. Furthermore, another activator of PKC, mezerein, inhibited the Ca2+ signal induced by OKT3. This inhibition, however, could completely be reversed by pretreatment with H-7 or staurosporine. We conclude that the TPAmediated inhibition of Ca2+ fluxes in Jurkat T cells largely acts through a PKCindependent pathway.
Proceedings of the National Academy of Sciences, 1988
We have investigated the linkage between CD4/CD8 phenotype and programing for specific responses in primary T-cell populations. In situ hybridization has been used to determine the frequency of cells competent to express the interleukin 2 (IL-2) gene after short-term stimulation with various polyclonal activators. The effects of the T-cell receptor ligands Con A and anti-CD3 monoclonal antibody were compared with those of a calcium ionophore that bypasses membrane receptors altogether. Induction with a calcium ionophore and phorbol ester revealed that potential IL-2 producers not only constitute >85% of the cells with a CD4' "helper/ inducer" phenotype but also constitute over half of the cells with a CD8' "killer/suppressor" phenotype. There is no defect in the ability of these CD8' cells to accumulate IL-2 transcripts under these conditions. By contrast, in response to phorbol ester and either Con A or anti-CD3, the CD8' cells show an abortive IL-2 production response with rapid disappearance of IL-2 mRNA. This results in substantially lower yields of IL-2 per cell than is made by CD4+ cells in response to the same stimuli. The extent to which these populations appear to have diverged in function thus depends on the stimulus used to trigger the response. The results suggest that differences in signal transduction or posttranscriptional regulatory mechanisms, rather than effector gene inducibility per se, may initially underlie the commitment of CD4+ and CD8+ cells to distinct functional roles. Mature T cells are functionally specialized in their responses to recognition of antigen. In general, T-cell lines that express the CD4 cell-surface glycoprotein are "helper" or "amplifier" cells that respond to the recognition of antigen by secreting lymphokines, often including the major T-cell growth factor interleukin 2 (IL-2) (1, 2). Cells that express CD8 include most killer T cells and show little helper activity (3-6). This suggests that the constitutive CD4/CD8 phenotypes of T cells are correlated with the inducibility of different, limited sets of functional response genes. Although the correlation is frequently observed in memory T cells and T-cell lines, it is not known how it is established during T-cell differentiation. When fresh CD8+ cells are activated and Abbreviations: IL-2, interleukin 2; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; nt, nucleotide.
International Journal of Immunopharmacology, 1991
Murine spleen cells, T-enriched by nylon wool filtration, proliferate in the presence of a protein kinase C stimulator and a calcium ionophore. Using this cell proliferation system, we show that LF 1695 can potentiate phorbol myristate acetate (PMA) action in the presence of A 23187. This potentiation can be due to PGE2 inhibition since it is found that lipopolysaccharide (LPS) or A 23187 induced PGE2 release from spleen cells is inhibited by LF 1695. Indomethacin and LF 1695 gave similar stimulation of spleen cell proliferation, and exogeneously added PGE2 inhibits this phenomenon. Considering two of the main early components of intracellular signal transduction, LF 1695 induces IP3 release and calcium mobilization. However, the compound is not mitogenic per se. These results show that LF 1695 behaves only as a costimulant for T-cell proliferation.