Activation of human T cells with the physiological regulator of protein kinase C

Purified resting human T cells can be induced to express the a subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-0-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the a subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.In several cell types, activation of cell responses results from the receptor-mediated hydrolysis of inositol phospholipids, generating two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, which activate protein kinase C (PKC) and elevate the intracellular Ca2+ concentration, respectively (reviewed in refs. 1 and 2). Antigenic activation of resting human T cells can be mimicked by the synergistic action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates PKC, and the calcium ionophore, ionomycin, which raises the intracellular Ca2+ concentration (3). This treatment results in the secretion of interleukin 2 (IL-2) and expression of a 55-kDa a subunit, which combines with a constitutively expressed 70-kDa ,B subunit (4, 5) to form the high-affinity IL-2 receptor (IL-2R; reviewed in ref. 6). Both expression of the a subunit of IL-2R (IL-2Ra) and secretion of IL-2 are controlled at the level of gene transcription (7) and represent a state of irreversible activation ofT cells, because autocrine binding of IL-2 to its cell surface receptor triggers proliferation (8).It was recently demonstrated that, in a >991% pure population of resting human T cells, replacing TPA with membrane-permeable DAGs, which activate PKC in a more physiological manner than TPA, does not induce expression ofthe IL-2Ra or cell proliferation (9). One explanation for the difference in cell response to TPA or DAGs is that TPA stimulates prolonged (longer than 1 hr) activation of PKC (10), whereas DAGs are rapidly converted to the corresponding phosphatidic acid by DAG kinase (11). In the present study, the duration of PKC activation by TPA or by the DAG 1,2-dioctanoylglycerol (DiC8) Chemical (Osaka), trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone was purchased from Worthington, and soybean trypsin inhibitor was from Sigma. MBP4_14 (GlnLys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu), synthesized in this laboratory, is a PKC-specific substrate (A.K. and Y.N., unpublished observations).Preparation of Pure T Lymphocytes. A population of resting T cells was purified from human peripheral ...

Section: Analysis Of Total Cellular Pkc Purified T Cells (25 X 107)mentioning

confidence: 98%

Activation of resting human T cells requires prolonged stimulation of protein kinase C.

Berry

Ase²,

Kishimoto³

et al. 1990

“…In the present studies, we examined these questions by use of two models of T-cell activation in which highly purified normal human T cells are induced to proliferate, in the absence of accessory cell signals, by signaling with a synergistic combination of monoclonal antibodies directed at the cluster designation 2 (CD2) and CD3 antigens expressed on the T-cell surface, or with a complementary combination of sn-1,2-dioctanoylglycerol (diC8) and ionomycin (22,23). The use of these T-cell activation systems permits evaluation of direct effects of GSH on T cells.…”

mentioning

confidence: 99%

“…The proliferative response is dependent on signaling T cells with both ionomycin and diC8 and is independent of additional antigenic or accessory signals (23). Ionomycin [Streptomyces conglobatus (ATCC31005); Calbiochem] and diC8 (Sigma) were dissolved in ethanol and then diluted with PBS (Sigma) to obtain the appropriate concentrations.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens.

Suthanthiran

Anderson

Sharma

et al. 1990

334

176

Regulation of proliferation of normal human T lymphocytes (T cells) by glutathione (GSH) was explored with T-cell activation models that-do not require accessory cell signals. L-Buthionine-(S,R)-sulfoximine (BSO), which inactivates y-glutamylcysteine synthetase and therefore inhibits GSH synthesis, inhibited proliferation elicited by monoclonal antibodies directed at cluster designation 2 (CD2) and CD3

“…Although the results reported from several laboratories are controversial (9,15,(18)(19)(20)(21), unlike PMA, a single dose of membrane-permeant DAG (such as 1,2-DiC8) is not fully sufficient to induce IL-2Ra expression. However, we found that the extent of IL-2Ra expression was dependent on the concentration of 1,2-DiC8 added (Table 1).…”

mentioning

confidence: 99%

Metabolic rate of membrane-permeant diacylglycerol and its relation to human resting T-lymphocyte activation.

Asaoka

Oka²,

Yoshida³

et al. 1991

DL-1,2-Dioctanoylglycerol (1,2-DiC8) added to human peripheral resting T lymphocytes was rapidly metabolized to produce octanoic acid and further to small molecules, probably by the action of diacylglycerol lipase and/or nonspecific esterase. Only a small portion was converted to the corresponding phosphatidic acid or was isomerized to 1,3-DiC8 before being metabolized. The uptake of 1,2-DiC8 by the cell was apparently fast, and the rate of disappearance of 1,2-DiCs was dependent on the cell densities; at a higher density of T lymphocytes 1,2-DiC8 was removed quickly, whereas at a lower cell density 1,2-DiC8 remained for a longer period oftime. With a fixed amount of 1,2-DiC8 added, the extent of interleukin 2 receptor a-subunit (IL-2Ra) expression was inversely related to the cell density and proportional to the duration of exposure of the cells to 1,2-DiCs. Repeated doses of 1,2-DiC8 potentiated IL-2Ra expression. In contrast, a single dose of phorbol 12-myristate 13-acetate caused T-lymphocyte activation to similr extents irrespective ofthe cell density, probably because the phorbol ester was not metabolized and remained in membranes. The available evidence supports a proposal made in a previous paper and indicates that the sustaind activation of protein kinase C for at least the first 3-4 hr is essential for the activation of resting T lymphocytes.Protein kinase C (PKC) is activated by diacylglycerol (DAG), which is produced physiologically as a result of the signalinduced hydrolysis of inositolphospholipids (1). Tumorpromoting phorbol esters and membrane-permeant DAGs such as 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol (DiC8) are often used to activate this enzyme within the intact cell (2, 3). Several discrepancies have been reported, however, between the responses induced by phorbol esters and those induced by the membrane-permeant DAGs (4-6). One possible explanation for the discrepancies is that the membrane-permeant DAG is metabolized quickly and activates PKC only transiently (2), whereas phorbol ester is hardly metabolized and thus activates the enzyme for a long time (7). Although the receptor-mediated hydrolysis of inositolphospholipids was once thought to be the sole mechanism leading to the activation of PKC (8), it is becoming plausible that the hydrolysis of inositolphospholipids is normally transient and insufficient to cause the sustained activation of PKC that appears to be needed for long-term responses such as cell proliferation and differentiation (9-11). For sustained activation of this enzyme, several mechanisms have been proposed, including phosphatidylcholine hydrolysis at a relatively later phase in cellular responses (12,13).In resting T lymphocytes, the synergistic action of phorbol ester and calcium ionophore has been reported to induce interleukin 2 (IL-2) secretion and IL-2 receptor a-subunit (IL-2Ra) gene expression, eventually leading to cell proliferation (for review, see ref. 14). A previous report from this laboratory (9) has shown that repeated doses of a...