Cyclolinteinone, a sesterterpene from sponge Cacospongia linteiformis, prevents inducible nitric oxide synthase and inducible cyclo-oxygenase protein expression by blocking nuclear factor-kappaB activation in J774 macrophages.
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ABSTRACT:
SUBMITTER: D'acquisto F
PROVIDER: S-EPMC1220914 | biostudies-other | 2000 Mar
REPOSITORIES: biostudies-other
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Project description:We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.
| S-EPMC1572810 | biostudies-other
Project description:Nitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1beta and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.Chondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1beta and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1beta and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data.IL-1beta activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1beta induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1beta induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1beta induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580.These data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.
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Project description:In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.
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Project description:Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance.
| S-EPMC5600347 | biostudies-literature
Project description:Here we report epigenomic and transcriptomic changes in a prototypical J774 macrophage after engulfing talc or titanium dioxide particles in presence of estrogen. Macrophages are the first immune cells to engage and clear particles of various nature. A novel paradigm is emerging, that exposure to so-called 'inert' particulates that are considered innocuous is not really free of consequences. We hypothesized that especially the insoluble, non-digestible particles that do not release a known hazardous chemical can be underappreciated agents acting to affect the regulation inside macrophages upon phagocytosis. We performed gene chip microarray profiling and found that talc alone, and especially with oestrogen, has induced a substantially more prominent gene expression change than titanium dioxide; the affected genes were involved in pathways of cell proliferation, immune response and regulation, and, unexpectedly, enzymes and proteins of epigenetic regulation. We therefore tested the DNA methylation profiles of these cells via epigenome-wide bisulphite sequencing and found vast epigenetic changes in hundreds of loci, remarkably after a very short exposure to particles; ELISA assay for methylcytosine levels determined the particles induced an overall decrease in DNA methylation. We found a few loci where both the transcriptional changes and epigenetic changes occurred in the pathways involving immune and inflammatory signalling. Some transcriptomic and epigenomic changes were shared between talc and titanium dioxide, however, it is especially interesting that each of the two particles of similar size and insoluble nature has also induced a specific pattern of gene expression and DNA methylation changes which we report here.
| S-EPMC8510616 | biostudies-literature
Project description:Obliterative bronchiolitis (OB) develops insidiously in nearly half of all lung transplant recipients. Although typically preceded by a CD8(+) T cell-rich lymphocytic bronchitis, it remains unresponsive to conventional immunosuppression. Using an airflow permissive model to study the role of gases flowing over the transplanted airway, it is shown that prolonged inhalation of sublethal doses of carbon monoxide (CO), but not nitric oxide (NO), obliterate the appearance of the obstructive airway lesion. Induction of the enzyme responsible for the synthesis of CO, heme oxygenase (Hmox) 1, increased carboxyhemoglobin levels and suppressed lymphocytic bronchitis and airway luminal occlusion after transplantation. In contrast, zinc protoporphyrin IX, a competitive inhibitor of Hmox, increased airway luminal occlusion. Compared with wild-type allografts, expression of inducible NO synthase (iNOS), which promotes the influx of cytoeffector leukocytes and airway graft rejection, was strikingly reduced by either enhanced expression of Hmox-1 or exogenous CO. Hmox-1/CO decreased nuclear factor (NF)-kappaB binding activity to the iNOS promoter region and iNOS expression. Inhibition of soluble guanylate cyclase did not interfere with the ability of CO to suppress OB, implicating a cyclic guanosine 3',5'-monophosphate-independent mechanism through which CO suppresses NF-kappaB, iNOS transcription, and OB. Prolonged CO inhalation represents a new immunosuppresive strategy to prevent OB.
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Project description:Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+](i)), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased ([Ca2+](i)) on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in ([Ca2+](i)) in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor kappaB (NF-kappaB) was investigated by electrophoretic mobility-shift assay. TG (100 nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1 ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100 ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100 nM) did not affect the NF-kappaB activity induced by low (1 ng/ml) or high (100 ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline ("XTT") method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1 microM) also transiently increased ([Ca2+](i)) and had opposite effects on NO production depending on the LPS concentration. Our results show that increased ([Ca2+](i)) induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in ([Ca2+](i)) might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.
| S-EPMC1221663 | biostudies-other
Project description:The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.
| S-EPMC1136387 | biostudies-other
Project description:Macrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis.The aim of this study is to understand whether heme oxygenase (HO) and nitric oxide synthase (NOS) contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and phagocytosis, two key components of macrophage function.This study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS) formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance.We show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme.Both enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.
| S-EPMC5786743 | biostudies-literature
Project description:Sustained phagocytosis requires the continuous replacement of cell-surface membrane from intracellular sources. Depending on the nature of the engulfed particles, a variety of endocytic compartments have been demonstrated to contribute membranes needed for the formation of phagosomes. It has recently been reported that the endoplasmic reticulum (ER) can also fuse with the plasma membrane during phagocytosis [Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J., Bergeron, J. J. & Desjardins, M. (2002) Cell 110, 119-131]. However, there is currently no known mechanistic basis for this fusion process to occur. Here we report that direct ER-plasma membrane fusion during phagocytosis requires the ER resident soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein ERS24/Sec22b and that J774-macrophages react toward the challenge of large (3.0-microm) but not small (0.8-microm) particles by triggering this fusion mechanism, allowing them to access the most abundant endogenous membrane source in the cell, the ER.
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