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S G Ward

Publications and source records attributed to S G Ward.

47 records · Page 3Linked to original sources
PubMed1995

Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin.

T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick, J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.

Androstadienes↗
PubMed1995

Both CD28 ligands CD80 (B7-1) and CD86 (B7-2) activate phosphatidylinositol 3-kinase, and wortmannin reveals heterogeneity in the regulation of T cell IL-2 secretion.

In this report, the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb. We demonstrate that while both anti-CD3 and anti-CD28 antibodies induced activation of phosphoinositide (PI) 3-kinase, the kinetics of activation differed. Anti-CD28 produced a sustained activation of PI 3-kinase while anti-CD3 induced activation was transient. Both B7-1 and B7-2 could induce prolonged activation of PI 3-kinase. The co-stimulatory effects of B7-1 and B7-2 were dependent on CD28 cross-linking, based on complete inhibition of PI 3-kinase activation by CD28 antibody Fab fragments. While Jurkat T cells co-stimulated with anti-CD3 and B7-1 or B7-2 secreted high levels of IL-2, there were distinct effects of anti-CD28 mAb and B7-1 or B7-2 on IL-2 secretion in conjunction with protein kinase C activation. To assess functional effects of CD28 ligation, pharmacologic inhibitors of PI 3-kinase were evaluated. In Jurkat cells, efficient inhibition of PI 3-kinase activation after B7-2 stimulation was achieved using wortmannin; however, we observed a surprising increase in IL-2 secretion after B7 or anti-CD28 stimulation. The effect of wortmannin was concentration dependent. Moreover, the effect was specific for receptor-mediated activation as wortmannin did not enhance phorbol ester plus ionomycin-induced IL-2 secretion. Another inhibitor of PI 3-kinase, LY294002, also resulted in augmentation of anti-CD28-induced IL-2 secretion by Jurkat cells. The effects of wortmannin on IL-2 secretion were also examined in primary T cells. In marked contrast, wortmannin resulted in a potent inhibition of anti-CD3 plus B7-1 or anti-CD28-induced IL-2 secretion while phorbol ester plus ionomycin-induced IL-2 secretion was wortmannin resistant. Together these observations demonstrate that signal transduction by both B7-1 and B7-2 involves PI 3-kinase, and that PI 3-kinase or other wortmannin-sensitive targets are important for IL-2 secretion. Finally, treatment of Jurkat cells with PI 3-kinase inhibitors alone was sufficient to induce low levels of IL-2 secretion. This is consistent with the notion that a wortmannin-sensitive target such as PI 3-kinase may down-regulate IL-2 secretion in Jurkat cells.

Androstadienes↗
PubMed1994

Calcium release activity and metabolism of inositol 1,4,5-trisphosphate in T cells. Modulation by novel inositol 1,4,5-trisphosphate 5-phosphatase inhibitors.

Stimulation of the T cell antigen receptor/CD3 complex is followed by phospholipase C activation, phosphoinositol lipid metabolism and ultimately by a rapid rise in both myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] as well as cytosolic free calcium concentration. A 5-phosphatase plays a pivotal role in the subsequent metabolism of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Synthetic routes have been developed which have enabled the synthesis of both natural and unnatural inositol phosphates and this approach has yielded several compounds which have been shown to act as inhibitors of Ins(1,4,5)P3 5-phosphatase. These compounds offer considerable potential for investigation of the complex metabolism and function of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in T cell activation and proliferation. We now report the time course and temperature sensitivity of Ins(1,4,5)P3-induced 45Ca2+ release in the permeabilised leukaemic T cell line Jurkat. Furthermore, we demonstrate that the metabolism of Ins(1,4,5)P3 in the presence of two novel 5-phosphatase inhibitors, namely L-myo-inositol 1,4,5-trisphosphorothioate [L-Ins(1,4,5)PS3] and myo-inositol 1,3,5-trisphosphorothioate [Ins(1,3,5)PS3], can be inhibited with concomitant elevation of the heparin-sensitive Ins(1,4,5)P3-induced release of 45Ca2+. These novel 5-phosphatase inhibitors provide a starting point for development of cell-permeable analogues which may be able to modulate cell function in intact cells and may be used as manipulative tools with which to elucidate the function of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 with respect to T cell activation.

Animals↗
PubMed1993

Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation.

The co-stimulatory role of B7/CD28 interactions is important in promoting T cell activation. Very little is known about the intracellular events that follow CD28 engagement although recent evidence has implicated coupling of CD28 to a protein tyrosine kinase signal transduction pathway. In this study we have investigated the putative role of D-3 phosphoinositides as mediators of CD28 receptor signaling, since phosphoinositide (PI) 3-kinase, the enzyme responsible for D-3 phosphoinositide formation, is a known substrate for protein tyrosine kinases associated with certain T cell surface receptors such as CD4 and interleukin-2 receptor. The lipid products of PI 3-kinase activity have been suggested to play a role in mitogenic signaling and growth regulation in other cells. Chinese hamster ovary cells (CHO) previously transfected with B7 cDNA, induced time-dependent elevation above basal levels of phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2) and PtdIns(3,4,5)P3, while parental CHO cells that did not express B7 had no effect on these lipids. Moreover, the elevation of these same lipids by CD3 ligation was potentiated in an additive manner by CHO-B7+ but not by CHO-B7- cells. CHO-B7+ and CHO-B7- cells did not activate phospholipase C as evidenced by their inability to modulate basal or CD3-induced changes in the levels of phosphatidic acid or D-4 and D-5 phosphoinositides. These data imply that PI 3-kinase but not phospholipase C, may be an important signal transduction molecule with respect to CD28-mediated co-stimulation and T cell activation following ligation by B7.

Animals↗
PubMed1992

Regulation of phosphoinositide kinases in T cells. Evidence that phosphatidylinositol 3-kinase is not a substrate for T cell antigen receptor-regulated tyrosine kinases.

A phosphoinositide kinase that can phosphorylate phosphatidylinositol (PtdIns) is present in 4G10 monoclonal antibody (mAb) phosphotyrosine immunoprecipitates isolated from T cells activated via the T cell antigen receptor (TCR).CD3 complex. This PtdIns kinase is not the PtdIns 3-kinase that associates with activated protein tyrosine kinases in fibroblasts, since Western blotting and immunoprecipitation experiments with antibodies specific for the p85 alpha subunit of the PtdIns 3-kinase indicate that this polypeptide is not immunoprecipitated by the 4G10 mAb from TCR.CD3-activated Jurkat cells. Moreover, immunoprecipitated PtdIns 3-kinase isolated from T cells with p85 antibodies is inhibited when PtdIns is presented in Nonidet P-40, whereas the PtdIns kinase activity present in 4G10 mAb phosphotyrosine immunoprecipitates is enhanced in the presence of Nonidet P-40. In vitro kinase assays of PtdIns 3-kinase immunoprecipitated with p85 antibodies from T cells indicate that it associates with a serine kinase that can phosphorylate a p85 polypeptide. However, no protein tyrosine kinase activity capable of tyrosine phosphorylating p85 in vitro associates with p85 alpha immunoprecipitates in quiescent or TCR.CD3-activated T cells. These data suggest that the TCR.CD3 complex does not regulate PtdIns 3-kinase activity by a mechanism that involves protein tyrosine kinases.

Adenosine↗
PubMed1992

Regulation of D-3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens.

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and the generation of inositol-(1,4,5)-trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D-4 and D-5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D-3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D-3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol-(3)-monophosphate [PtdIns(3)P], phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI3 kinase exist in T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes.

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Inhibition by staurosporine of mitogen-induced calcium mobilisation in human T lymphoblasts.

Addition of monoclonal antibodies (UCHT1) directed against the antigen receptor/CD3 complex or the addition of the mitogens concanavalin A or phytohemagglutinin to human T lymphoblasts induced an elevation of the cytosolic free calcium concentration [( Ca2+]i) as monitored by the fluorescence of the intracellular fura-2. The rise in [Ca2+]i induced by these agents was effectively inhibited by staurosporine, an agent which inhibits protein kinase (PK)C and PKA. Conversely the PKC activator 12-O-tetradecanoyl phorbol-13-acetate decreased the delay and accelerated the rate of elevation of [Ca2+]i induced by the mitogens and UCHT1. Increasing the concentration of cAMP or cGMP in the T lymphoblasts had no effect on agonist-induced elevation of [Ca2+]i. Our data indicate that PKC can exert a positive feedback on the level of cytosolic Ca2+ in T lymphocytes, in contrast to what has been observed with other cells.

Alkaloids↗
PubMed1988

Antagonism of the platelet activating factor-induced rise of the intracellular calcium ion concentration of U937 cells.

1. U937 cells are a continuous line of human cells of committed monocytic origin which serve as a useful model for studying human monocytic function. The present study investigated the effect of platelet-activating factor (Paf) on intracellular free calcium ion concentration ([Ca2+]i) in U937 cells using the calcium fluorescent probe fura-2. 2. The naturally-occurring stereoisomer (R)-Paf (0.01-300 nM) and the stable, less hydrolysable racemic Paf analogue PR1501 (10 nM-3 microM) produced dose-related and rapid elevations of 100-1200 nM [Ca2+]i above a basal value of 135 +/- 9 nM (n = 22). 3. The unnatural stereoisomer (S)-Paf and the natural stereoisomer lyso-(R)-Paf had no effect on basal [Ca2+]i at 30 microM, approximately 100,000 times the concentration found to be the threshold concentration to elicit a response to (R)-Paf. 4. Leukotriene B4 (LTB4) also induced increases in [Ca2+]i in the concentration range 28.5 nM-2.85 microM but the responses were smaller and of shorter duration than those induced by Paf. 5. Five compounds, WEB 2086, Ro 19-3704, L-652,731, BN 52021, and CV 3988, inhibited suboptimal Paf (10 nM)-induced increase in [Ca2+]i with IC50s of 48 +/- 2, 118 +/- 33, 318 +/- 131, 340 +/- 205 and 2320 +/- 183 nM respectively. All five compounds have previously been reported as specific Paf receptor antagonists, at least with respect to platelets. 6. The above compounds at 10 microM had no effect upon the increased [Ca2+]i induced by either LTB4 or the calcium ionophore, ionomycin. 7. These results suggest that U937 cells respond to Paf at least with respect to elevated [Ca2+]i as measured by fura-2 and that these cells may well possess a Paf receptor as suggested by the action of specific antagonists and the stereoselectivity observed with Paf.

Benzofurans↗
PubMed1987

The in-vitro and in-vivo efficacy of cisplatin and analogues in the treatment of herpes simplex virus-II infections.

The antitumour effect of cisplatin results from cross-linking and disruption of DNA when it binds to DNA bases, especially cytosine and guanine. Since herpes simplex virus (HSV) has a high cytosine and guanine content, cisplatin might be expected to have an antiviral effect against HSV. The 50% inhibitory concentration of cisplatin for HSV-II was 0.06 mg/l. Six of ten platinum analogues had 50% inhibition of plaques at less than or equal to 10 mg/l. We evaluated the in-vivo activity of cisplatin against the MS strain of HSV-II in the mouse genital HSV model. Mice were treated either intraperitoneally or intravaginally beginning at 3 or 51 h after inoculation. In the intraperitoneally treated groups infection rates were lower, but not significantly; 4 of 15 in the 3-h and 7 of 15-h group, compared to 9 of 15 in the untreated control group (P greater than 0.18, chi-square test). Intravaginal cisplatin demonstrated a significant reduction of the infection rate from 10 of 15 untreated controls, compared to 5 of 18 in the 3-h and 5 of 17 in the 51-h group (P less than 0.05, chi-square test). No toxic effects of intravaginal cisplatin were seen in uninfected mice. These studies suggest that platinum containing drugs warrant further evaluation as a new class of antiviral agents with activity against HSV.

Animals↗