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Biomedical subjects

S G Ward

Publications and source records attributed to S G Ward.

At least 19 recordsLinked to original sources
PubMed2001

Phosphoinositide 3-kinases in T lymphocyte activation.

Biochemical experiments have established that the metabolism of inositol phospholipids by phosphoinositide 3-kinases (PI3Ks) and lipid-phosphatases is triggered by many receptors that control T lymphocyte function, including antigen-receptors, costimulatory molecules, cytokines and chemokines. Novel effectors of PI3K have been identified in the immune system and shown to be important in the control of lymphocyte activation. Moreover, key lipid-phosphatases have been identified that act to terminate or modulate PI3K signalling in cells of the immune system.

Animals↗
PubMed2001

PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models.

Phosphoinositide 3-kinases (PI 3-Ks) phosphorylate the d3-hydroxyl position of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] resulting in the generation of the 3'-phosphoinositide lipid PtdIns(3,4,5)P(3). PI 3-Ks are activated by a diverse set of receptors that play a role in determining T-cell function. It now seems that leukemic T cells, which are widely used as models for T-cell biology, show constitutive activation of PI 3-K-mediated signal-transduction pathways. Hence, studies of the role of PI 3-K in T-cell biology using leukemic cell lines might have misinterpreted the importance of this pathway for T-cell signal transduction.

Enzyme Activation↗
PubMed2001

Phosphoinositide 3-kinases in the gut: a link between inflammation and cancer?

Carcinoma of the gastrointestinal tract is the most common internal malignancy affecting men and women in Western countries. Chronic intestinal inflammation, especially of the colon, is also a Western disease and correlates with a significantly increased risk of developing cancer. This has suggested that the immune processes involved in both conditions might share some common pathways. Indeed, there is increasing evidence that phosphatidylinositol 3-kinases (PI 3-kinases) are involved in both the pathogenesis of colorectal carcinoma and intestinal inflammation. Here, we discuss this rapidly progressing area of research, presenting evidence for a pivotal role of PI 3-kinase(s) in intestinal pathophysiology.

Animals↗
PubMed2001

Regulatory role of phosphatidylinositol 3-kinase on TNF-alpha-induced cyclooxygenase 2 expression in colonic epithelial cells.

BACKGROUND AND AIMS: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-alpha is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-alpha and its role in the regulation of genes controlled by NF-kappaB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. METHODS: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-alpha or interleukin (IL)-1alpha as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E(2) (PGE2) production by enzyme-linked immunosorbent assay. NF-kappaB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. RESULTS: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused up-regulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of < or =1 micromol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, down-regulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-kappaB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-alpha was shown to activate PI 3-kinase directly. CONCLUSIONS: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-13 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-kappaB because PI 3-kinase inhibition did not alter NF-kappaB binding activity. TNF-alpha can activate PI 3-kinase directly in addition to inducing COX-2.

Androstadienes↗
PubMed2001

The sheepshead minnow as an in vivo model for endocrine disruption in marine teleosts: a partial life-cycle test with 17alpha-ethynylestradiol.

The sheepshead minnow (Cyprinodon variegatus Lacépède), an estuarine fish species, was exposed to 17alpha-ethynylestradiol (EE2) at nominal test concentrations of 0.2, 2, 20, 200, 400, 800, 1,600, and 3,200 ng/L. Fish were exposed for up to 59 d, from subadult stages to sexual maturity, under flow-through conditions. The exposure period was followed by an evaluation of reproductive success and survival of progeny. The reproductive success of exposed sheepshead minnows, as determined from data on egg production from two subsequent spawning trials, was reduced in fish exposed to 200 ng/L EE2 and, in one spawning trial, in the 20-ng/L treatment. Hatching success was reduced in the progeny of fish exposed to 200 ng/L EE2, but survival was good among fry that successfully hatched. Histological examination indicated generalized edema, damage to gill epithelia, hepatic toxicity, fibrosis of the testis, and evidence of sex reversal, including testes-ova and spermatagonia-like cells in ovaries. The maximum acceptable toxicant concentration (MATC) for gonadal development in males was within the normal range of EE2 concentrations in sewage treatment plant effluents. The exposure regimen and choice of test organism, combined with histological examination, allowed independent evaluation of ecologically significant acute, reproductive and estrogenic endpoints. Estrogen receptor-mediated effects occurred at concentrations where reproductive effects were measurable under standard reproduction assays. The sheepshead minnow appears to be a sensitive in vivo model for partial life-cycle testing of compounds that have the potential to disrupt the endocrine system as well as reproduction in estuarine and coastal marine fish species.

Animals↗
PubMed2000

Interactions between phosphatidylinositol 3-kinase and nitric oxide: explaining the paradox.

Nitric oxide (NO) and the many derivatives and reactive oxygen intermediates thereof are all molecules that are utilised by mammalian cells in the war against microbial pathogens and tumours. They are potentially toxic molecules and, with damage control being crucial, the production and metabolism of nitric oxide is a tightly regulated process. The duality of NO is well documented. On the one hand, beneficial effects include normal healing in the skin and intestinal mucosa, killing of certain bacteria, regulating T cell proliferation and differentiation (Th1 vs Th2), and regulating leukocyte recruitment, by affecting adhesion molecule expression. On the other hand, persistent high levels of NO can lead to the production of toxic metabolites (peroxynitrite and hydroxyls), which can have detrimental effects, such as increased microvascular and epithelial permeability, increased oxidative stress (which can damage DNA), and damage to iron-sulphur proteins in mitochondria. NO has been reported to modulate its own production and the mechanisms involved in this self-regulation are being hotly pursued. The purpose of this review is to update recent intriguing advances in our understanding of the interaction of the phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathway in regulating the activity of the enzymes that generate NO, namely, the nitric oxide synthases.

Animals↗
PubMed2000

Phosphoinositide 3-kinase: a key biochemical signal for cell migration in response to chemokines.

Chemokines can couple to distinct signalling pathways that have been demonstrated to mediate not only migration, but also cell growth and transcriptional activation. One particular signalling pathway, namely that controlled by the lipid kinase phosphoinositide 3-kinase (PI3K), has been the focus of much attention with respect to its activation by chemokine receptors and the role it plays in regulating cell migration. Identification of PI3K is arguably one of the most exciting recent developments in biochemical signalling. Pharmacological and genetic studies have now convincingly shown that both CC and CXC chemokines stimulate PI3K-dependent chemotaxis of inflammatory cells such as eosinophils, macrophages, neutrophils and T lymphocytes. This review considers the role of specific sub-classes of PI3Ks (e.g. the p85/p110 heterodimer, PI3Kgamma and PI3K-C2alpha) as well as their downstream effector targets in mediating chemokine-stimulated cell migration.

Animals↗
PubMed2000

Disparate effects of phorbol esters, CD3 and the costimulatory receptors CD2 and CD28 on RANTES secretion by human T lymphocytes.

This study has examined the stimuli required for secretion of regulated upon activation, normal T-cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12-myristate 13-acetate (PMA), which are unable to support T-cell proliferation and interleukin-2 (IL-2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T-cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T-cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stimulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3-kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non-PKC activating 4alpha-phorbol ester, whilst the general PKC inhibitor Ro-32-0432 inhibits PMA-stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine-specific because PMA was unable to increase secretion of the related CC chemokine MIP-1alpha. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC-dependent pathways are sufficient for strong RANTES secretion, a calcium-dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte-associated antigen-4 (CTLA-4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA-stimulated RANTES secretion.

Abatacept↗
PubMed1999

The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes.

The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.

Actins↗
PubMed1999

Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation. Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism.

A combination of the pro-inflammatory cytokines interleukin (IL)-1alpha, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997) J. Biol. Chem. 272, 12626-12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-gamma/TNF-alpha, IFN-gamma/CD95, IL-1alpha/IFN-gamma, and IL-1alpha/IFN-gamma/TNF-alpha to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1alpha/IFN-gamma/TNF-alpha- and IFN-gamma/TNF-alpha- as well as IFN-gamma/CD95-induced (but not IL-1alpha/IFN-gamma-induced) cell death. In addition, IFN-gamma/TNF-alpha and IL-1alpha/IFN-gamma/TNF-alpha stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.

Amino Acid Sequence↗
PubMed1999

CD28 stimulates tyrosine phosphorylation, cellular redistribution and catalytic activity of the inositol lipid 5-phosphatase SHIP.

The D-3 phosphoinositide lipids phosphatidylinositol 3,4, 5-trisphophate [PtdIns(3,4,5)P(3)] and phosphatidylinositol 3, 4-bisphosphate [PtdIns(3,4)P(2)] represent upstream components of a major signaling pathway that is strongly activated by the T cell costimulatory molecule CD28. A major route for degradation of PtdIns(3,4,5)P(3) (and hence, regulation of PtdIns(3,4,5)P(3)-driven effector pathways), involves its conversion to PtdIns(3,4)P(2) by the 145-kDa SH2-containing inositol (poly)phosphate 5-phosphatase (SHIP). In this study, we demonstrate using the murine T cell hybridoma DC27.1, that SHIP is strongly tyrosine phosphorylated after ligation of CD28 by either mAb or the natural ligand B7.1. Ligation of CD3 also stimulates SHIP tyrosine phosphorylation and an additive effect on tyrosine phosphorylation of SHIP is observed when both CD3 and CD28 are ligated. The tyrosine phosphorylation of SHIP in response to CD28 ligation correlates with a marked redistribution of SHIP from the cytosol to the plasma membrane, as well as an increase in the in vitro 5-phosphatase activity associated with SHIP immunoprecipitates derived from CD28-stimulated cells. However, we have been unable to detect a direct association between CD28 and SHIP, so the mechanisms by which CD28 exerts the observed effects on SHIP remain unclear. This is the first demonstration that SHIP is a biochemical target for CD28 and suggests that SHIP may be involved in the regulation of T cell activation.

Animals↗
PubMed1999

The complexities of CD28 and CTLA-4 signalling: PI3K and beyond.

A successful immune response requires a set of non-cognate cell-cell interactions which provide the second "costimulatory" signal to the T cells. The best characterized costimulatory receptor expressed on resting T cells is CD28 which provides poorly-defined cyclosporin-resistant biochemical signal(s) that promote expression of several cytokines/chemokines. Another major effect of CD28 ligation is the promotion of cell survival which is thought to occur via the up-regulation of Bcl-xL expression, CD28 shares its ligands B7.1 and B7.2 with the related CTLA-4, which plays an inhibitory role in T cell activation. Manipulation of CD28/CTLA-4 interactions with their natural ligands has provided exciting results in transplantation and tumor therapy settings and also has potential in the treatment of several diseases such as arthritis and multiple sclerosis, asthma and protection against HIV infection. The biochemical basis for the different functional outcomes of CD28 and CTLA-4 ligation has been the subject of intense investigation over the past few years. This review will focus on our current understanding of the biochemical signals that may be involved in regulating the different functional outcomes of CD28 and CTLA-4, with particular emphasis on the role played by the PI3K-dependent signalling cascade.

Abatacept↗
PubMed1998

The CC chemokine monocyte chemotactic peptide-1 activates both the class I p85/p110 phosphatidylinositol 3-kinase and the class II PI3K-C2alpha.

The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 +/- 1.88 nM, n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 +/- 4.2 nM, n = 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2alpha) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2alpha activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2alpha rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2alpha. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2alpha enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.

Androstadienes↗
PubMed1998

Chemokines: understanding their role in T-lymphocyte biology.

The chemokines are a complex superfamily of small, secreted proteins that were initially characterized through their chemotactic effects on a variety of leucocytes. The superfamily is divided into families based on structural and genetic considerations and have been termed the CXC, CC, C and CX3C families. Chemokines from these families have a key role in the recruitment and function of T lymphocytes. Moreover, T lymphocytes have also been identified as a source of a number of chemokines. T lymphocytes also express most of the known CXC and CC chemokine receptors to an extent that depends on their state of activation/differentiation and/or the activating stimuli. The expression of two chemokine receptors, namely CXCR4 and CCR5, together with the regulated production of their respective ligands, appears to be extremely important in determining sensitivity of T cells to HIV-1 infection. The intracellular events which mediate the effects of chemokines, particularly those elicited by the CC chemokine RANTES, include activation of both G-protein- and protein tyrosine kinase-coupled signalling pathways. The present review describes our current understanding of the structure and expression of chemokines and their receptors, the effects of chemokines on T-cell function(s), the intracellular signalling pathways activated by chemokines and the role of certain chemokines and chemokine receptors in determining sensitivity to HIV-1 infection.

Chemokines↗
PubMed1997

Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.

B7-1 Antigen↗