Search PubMed⌕ Search

Biomedical subjects

L A O'Neill

Publications and source records attributed to L A O'Neill.

At least 19 recordsLinked to original sources
PubMed2001

The anti-inflammatory cytokine, interleukin (IL)-10, blocks the inhibitory effect of IL-1 beta on long term potentiation. A role for JNK.

Several effects of the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), have been described in the central nervous system, and one area of the brain where marked changes have been reported is the hippocampus. Among these changes are an IL-1 beta-induced inhibition of long term potentiation (LTP) in perforant path-granule cell synapses and an attenuation of glutamate release in synaptosomes prepared from the hippocampus. Evidence suggests that, at least in circulating cells, the anti-inflammatory cytokine, IL-10, antagonizes certain effects of IL-1. We investigated the effect of IL-10 on IL-1 beta-induced inhibition of LTP and glutamate release. The evidence presented indicates that IL-1 beta stimulates the stress-activated protein kinase, c-Jun-activated protein kinase (JNK), and IL-1 receptor-associated kinase, which may explain its inhibitory effect on release and LTP, and that IL-10 reversed the IL-1 beta-induced stimulation of JNK activity and inhibition of release and LTP. We observed that IL-10 abrogated the stimulatory effect of IL-1 beta on superoxide dismutase activity and reactive oxygen species production, whereas the H(2)O(2)-induced inhibition of LTP was also blocked by IL-10. We present evidence that suggests that the action of IL-10 may be mediated by its ability to induce shedding of the IL-1 type I receptor.

Animals↗
PubMed2001

The serine protease inhibitor antithrombin III inhibits LPS-mediated NF-kappaB activation by TLR-4.

In Drosophila, the Toll family of proteins mediates the innate immune response. Toll is activated by Spaetzle, which is generated in response to pathogens via a serine protease cascade. We wished to investigate if lipopolysaccharides (LPS) might activate Toll-like receptor (TLR) 4 via a serine protease in humans. The serpin antithrombin III (ATIII) and the thrombin inhibitor hirudin both inhibited nuclear factor (NF)-kappaB activation by LPS and Lipid A. ATIII and hirudin were also able to inhibit LPS-induced NF-kappaB activation in cells stably transfected with TLR4. These results suggest that LPS may activate a mammalian serine protease, which generates a product required for TLR4 signalling.

Antithrombin III↗
PubMed2001

Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt.

Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection.

Animals↗
PubMed2001

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The effects of the bacterial endotoxin lipopolysaccharide on synaptic transmission and plasticity in the CA1-subiculum pathway in vivo.

Lipopolysaccharide is derived from the cell wall of gram-negative bacteria and is a potent endotoxin which causes the release of cytokines in the CNS. We examined the effect of lipopolysaccharide on synaptic transmission and synaptic plasticity in the hippocampal area CA1-subicular pathway in vivo. We found that lipopolysaccharide did not affect baseline synaptic transmission in this pathway; it did, however, reduce the magnitude of paired-pulse facilitation, a form of short-term plasticity thought to be primarily presynaptic in origin. We then examined the interaction between lipopolysaccharide and two common models for the biological basis of memory: high-frequency stimulation induced long-term potentiation and low-frequency stimulation induced long-term depression of synaptic transmission. We found that lipopolysaccharide blocked long-term potentiation following high-frequency stimulation and also induced potentiation of synaptic transmission after low-frequency stimulation. Lipolysaccharide blocked paired-pulse facilitation selectively at short rather than longer interstimulus intervals. Thus, lipopolysaccharide has different effects on synaptic transmission in this pathway depending on the frequency and length of stimulation. These results provide new insights into the action of lipopolysaccharide on various forms of plasticity in the hippocampus, an area known to play a vital role in learning and memory.

Animals↗
PubMed2001

Transactivation by the p65 subunit of NF-kappaB in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1.

We have examined the involvement of components of the interleukin-1 (IL-1) signaling pathway in the transactivation of gene expression by the p65 subunit of NF-kappaB. Transient transfection of cells with plasmids encoding wild-type MyD88, IL-1 receptor-associated kinase 1 (IRAK-1), and TRAF-6 drove p65-mediated transactivation. In addition, dominant negative forms of MyD88, IRAK-1, and TRAF-6 inhibited the IL-1-induced response. In cells lacking MyD88 or IRAK-1, no effect of IL-1 was observed. Together, these results indicate that MyD88, IRAK-1, and TRAF-6 are important downstream regulators of IL-1-mediated p65 transactivation. We have previously shown that the low-molecular-weight G protein Rac1 is involved in this response. Constitutively active RacV12-mediated transactivation was not inhibited by dominant negative MyD88, while dominant negative RacN17 inhibited the MyD88-driven response, placing Rac1 downstream of MyD88 on this pathway. Dominant negative RacN17 inhibited wild-type IRAK-1- and TRAF-6-induced transactivation, and in turn, dominant negative IRAK-1 and TRAF-6 inhibited the RacV12-driven response, suggesting a mutual codependence of Rac1, IRAK-1, and TRAF-6 in regulating this pathway. Finally, Rac1 was found to associate with the receptor complex via interactions with both MyD88 and the IL-1 receptor accessory protein. A pathway emanating from MyD88 and involving IRAK-1, TRAF-6, and Rac1 is therefore involved in transactivation of gene expression by the p65 subunit of NF-kappaB in response to IL-1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase.

The transcription factor NF-kappaB is a central mediator of altered gene expression during inflammation, and is implicated in a number of pathologies, including cancer, atherosclerosis, and viral infection. We report in this study that vitamin C inhibits the activation of NF-kappaB by multiple stimuli, including IL-1 and TNF in the endothelial cell line ECV304 and in primary HUVECs. The induction of a NF-kappaB-dependent gene, IL-8, by TNF was also inhibited. The effect requires millimolar concentrations of vitamin C, which occur intracellularly in vivo, particularly during inflammation. Vitamin C was not toxic to cells, did not inhibit another inducible transcription factor, STAT1, and had no effect on the DNA binding of NF-kappaB. Inhibition by vitamin C was not simply an antioxidant effect, because redox-insensitive pathways to NF-kappaB were also blocked. Vitamin C was shown to block IL-1- and TNF-mediated degradation and phosphorylation of I-kappaBalpha (inhibitory protein that dissociates from NF-kappaB), due to inhibition of I-kappaB kinase (IKK) activation. Inhibition of TNF-driven IKK activation was mediated by p38 mitogen-activated protein kinase, because treatment of cells with vitamin C led to a rapid and sustained activation of p38, and the specific p38 inhibitor SB203580 reversed the inhibitory effect of vitamin C on IKK activity, I-kappaBalpha phosphorylation, and NF-kappaB activation. The results identify p38 as an intracellular target for high dose vitamin C.

Antioxidants↗
PubMed2000

A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling.

Poxviruses employ many strategies to evade and neutralize the host immune response. In this study, we have identified two vaccinia virus ORFs, termed A46R and A52R, that share amino acid sequence similarity with the Toll/IL-1 receptor (TIR) domain, a motif that defines the IL-1/Toll-like receptor (TLR) superfamily of receptors, which have a key role in innate immunity and inflammation. When expressed in mammalian cells, the protein products of both ORFs were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NFkappaB, and A52R potently blocked both IL-1- and TLR4-mediated NFkappaB activation. MyD88 is a TIR domain-containing adapter molecule known to have a central role in both IL-1 and TLR4 signaling. A52R mimicked the dominant-negative effect of a truncated version of MyD88 on IL-1, TLR4, and IL-18 signaling but had no effect on MyD88-independent signaling pathways. Therefore, A46R and A52R are likely to represent a mechanism used by vaccinia virus of suppressing TIR domain-dependent intracellular signaling.

Amino Acid Sequence↗
PubMed2000

Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells.

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.

Antigens, Neoplasm↗
PubMed2000

The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense.

The signal transduction pathways activated by the proinflammatory cytokine interleukin-1 (IL-1) have been the focus of much attention because of the important role that IL-1 plays in inflammatory diseases. A number of proteins have been described that participate in the post-receptor activation of the transcription factor NF-kappaB and stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK). It has also emerged that the type I IL-1 receptor (termed IL-1RI) is a member of an expanding receptor superfamily. These related receptors all have sequence similarity in their cytosolic regions. The family includes the Drosophila melanogaster protein Toll, the IL-18 receptor (IL-18R), and the Toll-like receptors TLR-2 and TLR-4, which bind molecules from Gram-positive and Gram-negative bacteria, respectively. Because of the similarity of IL-1RI to Toll, the conserved sequence in the cytosolic region of these proteins has been termed the Toll-IL-1 receptor (TIR) domain. The same proteins activated during signaling by IL-1RI also participate in signaling by IL-18R and TLR-4. The receptor superfamily is evolutionarily conserved; members occur in plants and insects and also function in host defense. The signaling proteins activated are also conserved across species. This receptor superfamily therefore represents an ancient signaling system that is a critical determinant of the innate immune and inflammatory responses.

Animals↗
PubMed2000

Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1.

We have found that lethal toxin from Clostridium sordellii, which specifically inactivates the low molecular weight G proteins Ras, Rap, and Rac, inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by interleukin-1 (IL-1) in EL4.NOB-1 cells and primary fibroblasts. The target protein involved appeared to be Ras, because transient transfections with dominant negative RasN17 inhibited p38 MAPK activation by IL-1. Furthermore, transfections of cells with constitutively active RasVHa-activated p38 MAPK. Further evidence for Ras involvement came from the observation that IL-1 caused a rapid activation of Ras in the cells and from the inhibitory effects of the Ras inhibitors manumycin A and damnacanthal. Toxin B from Clostridium difficile, which inactivates Rac, Cdc42, and Rho, was without effect. Dominant negative versions of Rac (RacN17) or Rap (Rap1AN17) did not inhibit the response. Intriguingly, transfection of cells with dominant negative Rap1AN17 activated p38 MAPK. Furthermore, constitutively active Rap1AV12 inhibited p38 MAPK activation by IL-1, consistent with Rap antagonizing Ras function. IL-1 also activated Rap in the cells, but with slower kinetics than Ras. Our studies therefore provide clear evidence using multiple approaches for Ras as a signaling component in the activation of p38 MAPK by IL-1, with Rap having an inhibitory effect.

Animals↗
PubMed2000

Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells.

We have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-kappa B. HA fragments activated NF-kappa B in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced I kappa B alpha phosphorylation and degradation, kappa B-linked reporter gene expression, and ICAM-1 promoter activity in an NF-kappa B-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7.8.1 blocked the effect on NF-kappa B. HA fragments activated the I kappa B kinase complex, and the effect on a kappa B-linked reporter gene was blocked in T-24 cells expressing dominant negative I kappa B kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-kappa B activation and I kappa B alpha phosphorylation. In particular, PKC zeta was required because transfection of cells with dominant negative PKC zeta blocked the effect of HA fragments on kappa B-linked gene expression and HA fragments increased PKC zeta activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-kappa B activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced kappa B-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade emanating from CD44 to Ras, PKC zeta, and I kappa B kinase 1 and 2.

Animals↗
PubMed2000

Rac1 regulates interleukin 1-induced nuclear factor kappaB activation in an inhibitory protein kappaBalpha-independent manner by enhancing the ability of the p65 subunit to transactivate gene expression.

We have examined the involvement of Rac1 in nuclear factor kappaB (NFkappaB) activation by interleukin 1 (IL1). IL1 induced a rapid and sustained activation of Rac1 in the thymoma cell line EL4.NOB-1. Transient transfection with dominant negative RacN17 inhibited IL1-induced kappaB-dependent reporter gene expression but not IkappaBalpha degradation, whereas constitutively active RacV12 potentiated kappaB-dependent reporter gene expression in response to IL1 but had no effects on its own. Using porcine aortic endothelial cells stably transfected with RacV12 or RacN17 under the control of an inducible promoter, we confirmed that RacV12 did not affect IkappaBalpha degradation, nor did RacN17 inhibit the IL1-induced response. RacV12 was also unable to induce nuclear translocation of NFkappaB. These effects suggested a role for Rac1 in p65-mediated transactivation of NFkappaB, independent of IkappaBalpha regulation. In support of this we found that IL1 activated a pathway leading to increased p65 transactivation activity and that RacV12 alone could drive this response in both cell systems. Additionally, RacN17 inhibited IL1-driven p65-mediated transactivation. From data using specific inhibitors of p38 and p42/p44 kinases we propose that both p38 and p42/p44 lie downstream of Rac1 on the IL1 pathway leading to enhanced transactivation by p65.

Humans↗
PubMed2000

Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries.

Nuclear factor-kappaB (NFKB) is a transcription factor with a pivotal role in inducing genes involved in physiological processes as well as in the response to injury and infection. A model has been proposed whereby the diverse agents that activate NFkappaB do so by increasing oxidative stress within the cell. Activation of NFkappaB involves the phosphorylation and subsequent degradation of an inhibitory protein, IKB, and recently many of the proximal kinases and adaptor molecules involved in this process have been elucidated. Additionally, we now understand in detail the NFkappaB activation pathway from cell membrane to nucleus for interleukin-1 (IL-1) and tumour necrosis factor (TNF). This review revisits the evidence for the oxidative stress model in light of these recent findings, and finds little in the new information to rationalise or justify a central role for oxidative stress in NF-kappaB activation. We demonstrate that much of the evidence for the involvement of oxidative stress is either specific to a stimulus in a particular cell line or open to reinterpretation. In particular, the activation of NFkappaB by hydrogen peroxide is cell-specific and distinct from physiological activators such as IL-1 and TNF, while inhibition by antioxidants, also found to be cell- and stimulus-specific, can involve diverse and unexpected targets which may be distinct from redox modulation. We conclude that in most cases the role of oxidative stress in NF-kappaB activation is at best facilitatory rather than causal, if a role exists at all. In addition, other evidence suggests a role for lipid peroxides in pathways where such a role exists. In future, when a role for oxidative stress in a pathway is postulated, the challenge will be to show which particular kinases or adaptor molecules, if any, are redox-modulated.

Animals↗
PubMed2000

The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products.

The interleukin-1 (IL-1) receptor/Toll-like receptor (TLR) superfamily is a recently defined and expanding group of receptors that participate in host responses to injury and infection. The superfamily is defined by the Toll/IL-1 receptor (TIR) domain, which occurs in the cytosolic region of family members, and is further subdivided into two groups based on homology to either the Type I IL-1 receptor or Drosophila Toll receptor extracellular domain. The former group includes the receptor for the important Th1 cytokine IL-18, and T1/ST2, which may have a role in Th2 cell function. The latter group includes six mammalian TLRs, including TLR2 and TLR4, that largely mediate the host response to gram-positive and gram-negative bacteria, respectively. Whether bacterial products are actual ligands for TLRs, or whether they generate ligands via as yet unidentified pattern recognition receptors, has yet to be determined. Signaling pathways activated via the TIR domain trigger the activation of downstream kinases, and transcription factors such as NF-kappaB, and involve the adaptor protein MyD88, which itself contains a TIR domain.

Animals↗
PubMed2000

The role of the interleukin-1/Toll-like receptor superfamily in inflammation and host defence.

The IL-1 receptor/Toll-like receptor superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions, termed the Toll/IL-1 receptor domain, which function in inflammation and host defence against microbial pathogens. Members include receptors for the proinflammatory cytokines IL-1 and IL-18 and Toll-like receptors 2 and 4, which are involved in host responses to Gram-positive and Gram-negative bacteria, respectively. Signalling pathways activated by these receptors are conserved and the superfamily represents a pan-genomic system involved in the host response to infection and injury.

Adaptor Proteins, Signal Transducing↗