Biomedical subjects

L Boscá

Publications and source records attributed to L Boscá.

At least 37 records · Page 2Linked to original sources

PubMed2000

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) inhibited the LPS- and IFN-gamma-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J(2), prostaglandin E(2), or rosiglitazone, a peroxisomal proliferator-activated receptor gamma ligand. Incubation of activated macrophages with 15dPGJ(2) inhibited the degradation of IkappaBalpha and IkappaBbeta and increased their levels in the nuclei. NF-kappaB activity, as well as the transcription of NF-kappaB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IkappaB phosphorylation showed an inhibition of IkappaB kinase by 15dPGJ(2) in cells treated with LPS and IFN-gamma, resulting in an impaired phosphorylation of IkappaBalpha, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IkappaB kinase with 2 microM 15dPGJ(2) reduced by 83% the phosphorylation in serine 32 of IkappaBalpha, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IkappaB kinase complex. These results show rapid actions of 15dPGJ(2), independent of peroxisomal proliferator receptor gamma activation, in macrophages challenged with low doses of LPS and IFN-gamma.

Animals↗

PubMed2000

Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis: involvement of phosphatidylinositol 3-kinase.

Challenge of Ins-1 cells, a rat beta-pancreatic cell line, with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) promoted the expression of type 2 nitric oxide synthase (NOS-2) in a cooperative way. Treatment of Ins-1 cells with IGF-I significantly inhibited the expression of NOS-2, especially at subsaturating concentrations of LPS and IFN-gamma. The inhibitory effect of IGF-I on NOS-2 expression was abrogated when cells were incubated with wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase. Transient expression of the p110 subunit of phosphatidylinositol 3-kinase impaired the LPS and IFN-gamma-dependent NOS-2 promoter activity in cells transfected with a 1-kb fragment corresponding to the 5'-flanking region of the NOS-2 gene. However, expression of a dominant negative form of p85 abolished the inhibitory action of IGF-I on the NOS-2 promoter activity. Analysis of the decreased NOS-2 promoter activity in cells incubated with IGF-I showed a lower nuclear factor KB binding as determined by electrophoretic mobility shift assays. The synthesis of NO, produced after LPS and IFN-gamma challenge, triggered an apoptotic response in these cells. IGF-I reduced apoptosis mainly through the decreased synthesis of NO. However, in activated cells treated with N-[3-(aminomethyl)benzyl]acetamidine, a specific NOS-2 inhibitor, IGF-I completely abolished the NO-independent apoptosis. This protection from apoptosis was dependent on phosphatidylinositol 3-kinase activity. These results suggest an important anti-inflammatory and anti-apoptotic role for IGF-I in beta-pancreatic cells, with both actions depending on the activation of phosphatidylinositol 3-kinase.

Animals↗

PubMed1999

Negative regulation by protein tyrosine phosphatase of IFN-gamma-dependent expression of inducible nitric oxide synthase.

Treatment of cultured peritoneal macrophages with IFN-gamma resulted in tyrosine phosphorylation of IkappaBalpha and IkappaBbeta, NF-kappaB activation, and expression of inducible NO synthase (iNOS). Since tyrosine phosphorylation of IkappaBalpha is sufficient to activate NF-kappaB in Jurkat cells, macrophages were treated with the protein tyrosine phosphatase inhibitor peroxovanadate (POV), which elicited an intense tyrosine phosphorylation of both IkappaB. However, this phosphorylation failed to activate NF-kappaB. Treatment with POV of macrophages stimulated with IFN-gamma or LPS potentiated the degradation of IkappaBalpha and IkappaBbeta, the activation of NF-kappaB, and the expression of iNOS. Analysis of the iNOS gene promoter activity corresponding to the 5'-flanking region indicated that POV potentiates the cooperation between IFN-gamma-activated transcription factors and NF-kappaB. These results indicate that tyrosine phosphorylation of IkappaB is not sufficient to activate NF-kappaB in macrophages and propose a negative role for protein tyrosine phosphatase in the expression of iNOS in response to IFN-gamma.

Adjuvants, Immunologic↗

PubMed1999

Negative regulation by phosphatidylinositol 3-kinase of inducible nitric oxide synthase expression in macrophages.

Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase (PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promoted a prolonged activation of NF-kappaB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from transfection experiments using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Cotransfection of cells with a catalytically active p110 subunit of PI3-kinase impaired the responsiveness of the iNOS promoter to LPS stimulation, whereas transfection with a kinase-deficient mutant of p110 maintained the up-regulation in response to wortmannin. These results indicate that PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the mechanism of action of antiinflammatory cytokines.

Androstadienes↗

PubMed1999

Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators.

Programmed cell death occurs in several physiopathological situations in multicellular organisms and constitutes a common mechanism of cell replacement, tissue remodelling and removal of altered cells. The effectors that induce apoptosis as well as the signalling pathways involved in the process are the subjects of current work. In addition to receptor-mediated apoptosis, highly reactive molecules, such as NO, influence cell viability either by acting as a protection against apoptogenic stimuli, or by inducing apoptosis when produced at elevated concentrations. The contribution to apoptosis of mediators released by the mitochondria and involved in the activation of caspases focused attention on the functional changes caused by NO in this organelle. NO induces mitochondrial permeability transition and promotes apoptosis in cell-free systems containing mitochondria and nuclei. Moreover, NO-dependent apoptosis can be blocked in most cases through the use of permeability transition or caspase inhibitors. The intracellular pathways activated in response to NO challenge and involved in the regulation of apoptosis are analysed.

Animals↗

PubMed1999

Presence of a nitric oxide synthase inhibitor in the graft efflux during reperfusion in human liver transplantation.

Involvement of the nitric oxide (NO) system in complications following human orthotopic liver transplants (OLT) has been reported, but the contribution of the graft to the modulation of the NO system during reperfusion in normal OLT has not been characterized. We have studied the contribution of the graft efflux to the modulation of the NO system in 20 consecutive OLT. We evaluated its effects on isolated vascular reactivity of the rabbit and on rat cultured macrophages stimulated with lipopolysaccharide (LPS). In none of the donor liver biopsies was expression of inducible NO synthase (iNOS) activity by Northern or Western blot analysis found. Graft efflux after the onset of liver reperfusion, but not pre-transplant patient plasma, reversibly inhibited the acetylcholine-induced relaxation of norepinephrine-contracted rabbit aortic rings. Moreover, graft efflux reversibly inhibited NO production in rat macrophages treated with LPS, as evidenced by both a decrease in nitrite plus nitrate formation and a decrease in the production of [14C]citrulline from [14C]arginine. Addition of a 10% dilution of graft efflux to cultured rat macrophages incubated with LPS increased iNOS mRNA levels, suggesting direct inhibition of the enzyme but not of its expression. These results cannot be ascribed to the depletion of arginine the iNOS substrate since they can be reproduced even in the presence of an excess (10 mM) of exogenously added arginine. No correlation was found between the iNOS inhibitory activity in each sample and the corresponding clinical parameters related to either the graft function after the OLT or the existence of post-reperfusion syndrome. Our results indicate the existence of a soluble factor in the graft efflux from human OLT that reversibly and unspecifically inhibits NOS activity. Its involvement in the physiology and/or pathology of human liver diseases deserves further study.

Acetylcholine↗

PubMed1999

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages.

1. Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon-gamma (IFN-gamma) induced apoptotic death through a nitric oxide-dependent pathway. 2. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation-dependent apoptosis. 3. These drugs decreased the up-regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl-2 than in LPS/IFN-gamma treated cells. 4. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. 5. Incubation of macrophages with LPS/IFN-gamma barely affected caspase-1 but promoted an important activation of caspase-3. Both CsA and FK506 inhibited pathways leading to caspase-3 activation. Moreover, the cleavage of poly(ADP-ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. 6. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase-3 in cells treated with nitric oxide donors. 7. These results indicate that CsA and FK506 protect macrophages from nitric oxide-dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.

Animals↗

PubMed1999

Inhibition of NOS-2 expression in macrophages through the inactivation of NF-kappaB by andalusol.

1. Andalusol, ent-6alpha,8alpha,18-trihydroxy-13(16),14-labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844-849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). 2. Incubation of J774 macrophages with andalusol (0.1 - 100 microM) inhibited the synthesis of nitrite caused by LPS (1 microg ml-1) in concentration and time-dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h. 3. Incubation of J774 cells with LPS resulted in the expression of NOS-2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme decreased significantly in the presence of andalusol (IC50=10.5 microM), suggesting that this diterpene inhibited NOS-2 expression. 4. Andalusol inhibited nuclear factor kappaB activation, a transcription factor necessary for NOS-2 expression in response to LPS and IFN-gamma. This compound also inhibited the degradation of IkappaBalpha favouring the retention of the inactive NF-kappaB complexes in the cytosol. 5. Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS-2 expression in LPS-activated macrophages. The present findings provide a mechanism by which the anti-inflammatory properties of this diterpene could be mediated.

Animals↗

PubMed1999

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices.

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.

Animals↗

PubMed1999

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Animals↗

PubMed1999

Requirement of nuclear factor kappaB for the constitutive expression of nitric oxide synthase-2 and cyclooxygenase-2 in rat trophoblasts.

Recently isolated trophoblasts express nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2), decreasing the levels of the corresponding mRNAs when the cells were maintained in culture. The sustained expression of COX-2 and NOS-2 in trophoblasts was dependent on the activation of nuclear factor kappaB (NF-kappaB) since proteasome inhibitors and antioxidants that abrogated NF-kappaB activity suppressed the induction of both genes. The time-dependent fall of the mRNA levels of NOS-2 and COX-2 paralleled the inhibition of NF-kappaB, determined by electrophoretic mobility shift assays, and the increase of the IkappaBalpha and IkappaBbeta inhibitory proteins. Isolated trophoblasts synthesized reactive oxygen intermediates (ROI), a process impaired after culturing the cells, and that might be involved in the NF-kappaB activation process. Moreover, treatment of recently isolated cells with ROI scavengers suppressed the expression of COX-2 and NOS-2. Challenge of trophoblasts with interleukin-1beta up-regulated the expression of both proteins, an effect that was potentiated by lipopolysaccharide. These results indicate that the physiological expression of NOS-2 and COX-2 in trophoblasts involves a sustained activation of NF-kappaB which inhibition abrogates the inducibility of both genes.

Animals↗

PubMed1999

Inhibition of prostaglandin synthesis up-regulates cyclooxygenase-2 induced by lipopolysaccharide and peroxisomal proliferators.

Primary cultures of fetal hepatocytes expressed cyclooxygenase-2 (COX-2) upon stimulation with bacterial lipopolysaccharide (LPS) or peroxisomal proliferators. This enzyme was active and a good correlation between the mRNA levels, the amount of protein, and the synthesis of prostaglandin E2 was observed. However, when cells were incubated in the presence of indomethacin or the COX-2-specific inhibitor NS398, the amount of COX-2 protein increased 5-fold after activation with LPS and 2-fold after treatment with clofibrate. This up-regulation of COX-2 was not observed at the mRNA level. The mechanism of protein accumulation might involve either a direct stabilization of the enzyme by the inhibitors or the absence of prostaglandins involved in the regulation of its turnover. Among the prostaglandins assayed, only 15-deoxy-Prostaglandin J2 exerted a statistically significant decrease in the COX-2 levels in cells stimulated with LPS or LPS plus NS398. The accumulation of COX-2 in the presence of inhibitors was also observed in peritoneal macrophages treated under identical conditions. These results indicate that COX-2 protein accumulates after enzyme inhibition, and because removal of the inhibitors restored the enzyme activity, suppression of treatment with reversible COX-2 inhibitors may cause a transient overproduction of prostaglandins.

Animals↗

PubMed1998

Suppression of HIV-1 infection in linomide-treated SCID-hu-PBL mice.

BACKGROUND: Proinflammatory cytokine overproduction, as well as synthesis of the inducible form of nitric oxide synthase (iNOS), are known to play a major role in HIV-1-triggered disease. AIDS patients show increased serum tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels, which synergize with HIV-1-produced nitric oxide (NO) to augment viral replication. Linomide has strong immunomodulatory effects in animals and humans, yielding promising clinical benefits in several pathological disorders including septic shock and autoimmune disease, processes largely mediated by overproduction of these cytokines. In peripheral T cells, linomide also prevents apoptosis triggered by a variety of stimuli, including superantigens, dexamethasone and vaccinia virus. DESIGN AND METHODS: Linomide inhibits production of proinflammatory cytokines such as TNF-alpha, interleukin-1 beta and IFN-gamma, as well as iNOS synthesis. The SCID-hu-PBL mouse model was used to analyse the effect of linomide on HIV-1 infection. T-cell frequency was characterized in reconstituted animals, and the frequency of infected mice and viral load of infected animals were studied. RESULTS: Linomide promotes an increase in human CD4+ T-cell counts in the peritoneal cavity of HIV-1-infected, linomide-treated mice. Linomide also prevents human TNF-alpha and IFN-gamma production, as well as iNOS expression and affects the viral load, promoting potent suppression of HIV-1 infectivity as detected in peritoneal cavity and spleen. CONCLUSIONS: The combination of linomide's properties, namely, blockage of proinflammatory cytokine and NO production, as well as prevention of apoptosis, is of paramount interest, making linomide a potential candidate for combating HIV-1 infection or preventing some of its associated pathological manifestations.

Adjuvants, Immunologic↗

PubMed1998

Triggering of peritoneal macrophages with IFN-alpha/beta attenuates the expression of inducible nitric oxide synthase through a decrease in NF-kappaB activation.

Triggering peritoneal macrophages with IFN-gamma and a low concentration of LPS induced the expression of the inducible form of nitric oxide synthase (iNOS). This process was significantly inhibited when IFN-alpha/beta was added during the initial 2 h after the start of IFN-gamma/LPS activation. Evaluation of the transcriptional activity using run-on assays indicated that IFN-alpha/beta inhibited the transcription of iNOS. Transfection experiments using a 1.7-kb promoter sequence corresponding to the 5' flanking region of the murine iNOS gene showed decreased promoter activity in the presence of type I IFNs. Analysis of the transcription factors that participate in iNOS expression revealed a marked decrease of NF-kappaB activation, a nuclear factor required for the transcription of this gene. The degradation of IkappaB alpha and IkappaB beta, which is required for the translocation of NF-kappaB to the nucleus, was inhibited in the presence of IFN-alpha/beta. However, the activity of other transcription factors such as IFN regulatory factor 1, which is involved in the expression of iNOS in response to IFN-gamma, was not affected by IFN-alpha/beta stimulation. These results suggest that in the presence of IFN-alpha/beta, the activity of the iNOS promoter is impaired, and this attenuated nitric oxide synthase expression could be important in pathophysiologic situations in which secretion of type I IFNs occurs.

Animals↗

PubMed1998

Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines.

Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of inflammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-inflammatory cytokines. Since hepatocytes respond to LPS and pro-inflammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-alpha and interleukin-1beta. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E2, respectively. The use of NS 398, a specific pharmacological inhibitor of COX-2, confirmed the expression of this isoenzyme in activated foetal hepatocytes. Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-alpha and interleukin-1beta. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-beta inhibited the LPS- and pro-inflammatory cytokines-dependent expression of COX-2. These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-inflammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Aging↗

PubMed1998

Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices.

Nitric oxide (NO) overproduction has been postulated to contribute significantly to ischaemia-reperfusion neurotoxicity. Inducible or type II NO synthase (iNOS) synthesizes NO in large quantities for long periods of time. Therefore we investigated the expression and localization of iNOS after oxygen and glucose deprivation in rat forebrain slices. In this experimental model, calcium-independent NOS activity reached a maximum 180 min after the end of a 20 min oxygen-glucose deprivation period. During the same period of time, the calcium-independent activity was absent in control forebrain slices. To test whether this calcium-independent NOS activity was due to the expression of iNOS, the effects of the addition of dexamethasone, cycloheximide and pyrrolidine dithiocarbamate were determined. All of them inhibited the induction of the calcium-independent NOS activity measured in the rat forebrain slices after oxygen and glucose deprivation. Furthermore, oxygen and glucose deprivation caused the expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message and protein in these samples. A sixfold increase in the iNOS mRNA levels was observed at 180 min and the time-course of the expression of iNOS mRNA was in agreement with the temporal profile of iNOS enzymatic activity. Immunohistochemistry analysis revealed that iNOS was highly expressed in neurones, astrocytes and microglial cells. These results demonstrate for the first time that iNOS is expressed in neurones after oxygen and glucose deprivation, and that this expression occurs in short periods of time. These findings suggest that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia.

Animals↗

PubMed1998

The c-erbA alpha protooncogene induces apoptosis in glial cells via a protein kinase C- and bcl-2-suppressible mechanism.

The c-erbA protooncogene encodes the thyroid hormone (3,5,3'-triiodothyronine; T3) receptor alpha1 (TR alpha1). c-erbA/TR alpha1 is expressed in many cell types including glial cells, particularly in the immature state. We show here by morphological and biochemical criteria that c-erbA induces apoptosis of glial B3.1 cells in serum-deprived conditions. This effect is mostly T3 independent. Growth factors such as platelet-derived growth factor, basic fibroblast growth factor, or transforming growth factor-alpha prevent B3.1 + TR alpha1 cell death. Protein kinase C (PKC) activators also prevent the apoptosis phenomenon, an effect that was blocked by the PKC-specific inhibitor GF109203X. Expression of an exogenous bcl-2 gene led also to B3.1 + TR alpha1 cell survival. Neither a series of inhibitors including GF109203X nor T3 inhibits bcl-2 action, indicating that bcl-2 blocks a downstream step in the death-promoting process. B3.1 + TR alpha1 cell apoptosis is not blocked by caspase-1 or poly-ADP-ribosyltransferase inhibitors, suggesting that the activation of these classic pathways is not involved in the apoptotic mechanism. In addition, direct interaction with specific neuronal cells but not incubation with their conditioned medium inhibits also apoptosis of B3.1 + TR alpha1 cells. Our results show that c-erbA promotes an apoptotic process in glial B3.1 cells that is suppressible by PKC activation and bcl-2, probably by distinct mechanisms.

Animals↗

PubMed1998

Interferon-alpha/beta inhibits the apoptosis induced by lipopolysaccharide and interferon-gamma in murine peritoneal macrophages.

Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-alpha/beta) down-regulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-gamma. Moreover, IFN-alpha/beta also decreased cell death in LPS/IFN-gamma-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-alpha/beta) against the apoptosis induced by treatment with LPS and IFN-gamma showed that type I IFNs were very effective when added up to 1 h after IFN-gamma/LPS stimulation. Addition of IFN-alpha/beta 4 h after stimulation with IFN-gamma/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-alpha/beta suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections.

Animals↗