Henning Dr Sommermeyer

Pertussis toxin B-subunit-induced Ca2+-fluxes in Jurkat human lymphoma cells: The action on long-term pre-treatment with cholera and pertussis holotoxins

The exotoxins of Bordetella pertussis and Vibrio cholera have been used to investigate signal transduction in the human T-cell lymphoma Jurkat. Stimulation of the cells, leading to an increase in cytoplasmic free calcium, could be achieved by the anti-T-cell receptor complex antibody OKT3 and by pertussis holotoxin (PTHT), or its B-subunit (PTB), but not by cholera holotoxin (CTHT) or its B-subunit (CTB). Both holotoxins ADP-ribosylated specifically G-proteins in the plasma membrane of intact cells, while their B-subunits had no ADP-ribosyltransferase activity. Incubation of the cells with CTHT led to a state of unresponsiveness to all stimulants. CTB was without any effect, indicating that the ADP-ribosyltransferase activity of cholera toxin (located in the A-subunit of the holotoxin) was necessary for the inhibition of cellular signalling. The inhibitory effect of cholera toxin on the pertussis toxin action was not due to a blockade of pertussis toxin interaction with the cell surface, because pertussis toxin was still able to ADP-ribosylate membrane proteins in cholera toxin treated intact cells. In addition, the cholera toxin mediated inhibition was not due to elevated levels of cyclic-AMP, as forskolin (a direct activator of the adenylate cyclase) and no inhibitory effect. The stimulating effect of PTHT was independent of its ADP-ribosyltransferase activity, because it could also be obtained by the B-subunit alone. In addition, the increase of cytoplasmic free calcium after stimulation by PTHT clearly preceded the ADP-ribosylation. Pre-treatment with PTHT, PTB or OKT3, led to a long lasting increase in the level of intracellular Ca2+ in Jurkat cells, which could not, therefore, be stimulated further. Inhibition by cholera holotoxin of the stimulation by OKT3 and pertussis toxin (PTHT and PTB) imply that the mitogenic effect of pertussis toxin is perhaps mediated via the T-cell antigen receptor signalling cascade. The presented data do not support the idea that a pertussis toxin-sensitive G-protein is involved in coupling the T-cell antigen receptor to the phospholipase C.

Transforming growth factors β1 and β2 as well as milk growth factor decrease anti‐CD3‐induced proliferation of human lymphocytes without inhibiting the anti‐CD3‐mediated increase of [Ca2+]i and the activation of protein kinase C

Porcine transforming growth factor 1 and 2 (pTGF‐β1 and ‐β2) and milk growth factor (MGF) at 1 ng/ml significantly inhibited the proliferation of human lymphocytes induced by anti‐CD3 antibodies. In contrast, the anti‐CD3‐mediated increase of intracellular Ca2+ and the activation and translocation of protein kinase C were not affected by the transforming growth factors.

Activation of human thymocytes by antibodies to the CD3/T-cell receptor complex: Triggering of different epitopes results in different signals☆

Monoclonal antibodies (mAb) against the CD3/T cell receptor (TcR) complex were analyzed for their ability to activate human thymocytes. In addition to mAb detecting epitopes on the CD3 complex (OKT3, BMA 030) the activation potential of recently developed mAb against common epitopes on the alpha/beta T-cell receptor (anti-TcR mAb: BMA 031, BMA 032) was evaluated. Several differences were observed between the two types of mAb: (a) Binding of the tested anti-CD3 mAb to thymocytes resulted in a rapid increase in the level of cytoplasmic free calcium ions [Ca2+]i, whereas no significant changes in [Ca2+]i were detected in thymocytes stimulated with BMA 031 or BMA 032. (b) Induction of effective proliferation induced by mAb OKT3 depended on exogenous IL-2 and in addition on the presence of accessory cells or phorbol-ester. Proliferation induced by BMA 031 only required exogenous IL-2. (c) OKT3 but not BMA 031 inhibited proliferation of thymocytes induced via the CD2 molecule. These studies indicate that anti-CD3 and anti-TcR mAb transduce different signals in thymocytes. Since the two types of mAb are directed to the same molecular complex the observed differences also support the idea that there are functionally different compartments in the CD3/TcR complex which may activate different signaling pathways.

Cholera Toxin-Mediated Inhibition of Signalling in Jurkat Cells is followed by, but not Due to a Loss of T Cell Receptor Complex

Cholera toxin treatment of the human T cell lymphoma Jurkat resulted in inhibition of signalling via the T cell antigen receptor complex (TcR/CD3-complex). Cholera toxin specifically ADP-ribosylated the alpha-subunit of the stimulatory G-protein of the adenylate cyclase (Gs alpha), no other proteins were modified in the intact cells. ADP-ribosylation of Gs alpha and its subsequent activation led to an increase of the cyclic AMP level and in addition, to a drastic reduction of the cell-surface density of the TcR/CD3-complex. Recently, we demonstrated that the effect of cholera toxin at the receptor level is not due to an increased cAMP level (4). As inhibition of signalling is also not cAMP-mediated (8), we examined whether the modulation of the TcR/CD3-complex could be the reason for the interruption of the signalling cascade. Analyzing the time courses of the multiple cholera toxin effects in Jurkat cells at 37 degrees C, the following sequence was found: ADP-ribosylation of Gs alpha--increase of cyclic AMP level--inhibition of signalling via the TcR/CD3-complex--decrease of cell-surface density of the TcR/CD3-complex. Treatment of Jurkat cells at 20 degrees C with cholera toxin resulted in an increase of cyclic AMP and inhibition of signal transduction, while no decrease of TcR/CD3-complex density could be observed. These data imply that receptor loss from the cell-surface is not causative for the inhibition of signalling. More likely, activation of Gs uncouples signal transduction in Jurkat cells via the TcR, which by a so far unknown mechanism is followed by a loss of the receptor from the cell surface.

Signalling via rat dopamine D2-receptors expressed in mouse fibroblasts is not influenced by compounds binding to the sigma sites of these cells

The mouse fibroblast cell line LZR-1 is a well-established test system used to characterize the intrinsic activity of dopamine D2-receptor ligands (Neve et al., Mol. Pharmac., 1989). This cell line is transfected with a eucaryotic expression vector containing the gene of the rat dopamine D2B-receptor (short isoform of the D2-receptor) (Bunzow et al., Nature, 1988). In addition to the expression of high levels of the rat dopamine D2-receptor, these mouse cells also express sigma binding sites. Binding affinities of BMY 14,802, DTG, haloperidol, (+)-pentazocine, (+)-3-PPP, and (+)-SKF 10,047 for the sigma binding sites as well as for the D2-dopamine receptor in membranes of LZR-1 cells were determined. Using intact LZR-1 cells, it was found that the influence of sigma ligands on signalling via the dopamine D2-receptor can be explained by their affinity for the latter receptor. Specific sigma ligands did not influence dopaminergic signal transduction in LZR-1 cells. It is therefore concluded that the LZR-1 cell is a suitable test model for determination of the intrinsic activity of dopamine D2-receptor ligands even if these compounds have affinity for sigma binding sites.

Eicosanoid Synthesis in Resident Macrophages: Role of Protein Kinase C

In this study we investigated the effects of different agents known as activators or inhibitors of the calcium and phospholipid-dependent protein kinase (PKC) on the eicosanoid production in resident mouse peritoneal macrophages and in the murine macrophage-like cell line HA38. PKC activators such as the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA) or the synthetic cell-permeable diacylglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) stimulated arachidonic acid (AA) release from prelabelled cells and also decreased AA incorporation into cellular phospholipids within minutes. This rapid enlargement in the amount of free AA available for eicosanoid synthesis resulted in a significantly enhanced cellular prostaglandin E2 (PGE2) production, whereas leukotriene C4 (LTC4) could not be detected under these experimental conditions. Increasing the intracellular calcium level by simultaneous addition of calcium ionophores together with PKC activators in suboptimal concentrations led to a synergistic production of PGE2 and LTC4. PKC inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), sphingosine, tamoxifen, and staurosporine, which exert their inhibitory action on PKC by different mechanisms, totally abolished TPA effects, whereas changes induced by (DiC8) or OAG were only partially reversed. Aluminum fluoride (AlF4 −) as an activator of regulatory guanine nucleotide binding proteins (G-proteins) elevated the intracellular concentration of inositolphosphates (IP) and diacylglycerols (DAG), thus leading to an activation of PKC and an increase in the intracellular calcium level, and subsequent enhanced eicosanoid production. AlF4 −-mediated PGEz and LTC4 synthesis again was reversed by using PKC inhibitors. Preincubation of the cells with pertussis toxin (PT) did not inhibit but even enhanced PGE2 production stimulated by zymosan or AlF4 −. Depletion of PKC activity by prolonged incubation of macrophages with TPA resulted in an extensive abrogation of the A1F4 − or zymosan-induced PGE2 secretion. These data provide further evidence that PKC is centrally involved in the regulation of macrophage eicosanoid synthesis initiated by different stimuli.