Elizabeth Pintado

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5′‐AMP‐activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5′‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) or with 5′‐AMP significantly attenuated both phorbol 12‐myristate 13‐acetate (PMA) and formyl methionyl leucyl phenylalanine‐stimulated superoxide anion (O2 −) release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA‐dependent H2O2 release, and induced the phosphorylation of c‐jun N‐terminal kinase 1 (p46), p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5′‐AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.

Emerging topics in FXTAS

This paper summarizes key emerging issues in fragile X-associated tremor/ataxia syndrome (FXTAS) as presented at the First International Conference on the FMR1 Premutation: Basic Mechanisms & Clinical Involvement in 2013.

Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome

Carriers of the fragile X premutation (FPM) have CGG trinucleotide repeat expansions of between 55 and 200 in the 5′-UTR of FMR1, compared to a CGG repeat length of between 5 and 54 for the general population. Carriers were once thought to be without symptoms, but it is now recognized that they can develop a variety of early neurological symptoms as well as being at risk for developing the late onset neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). Several mouse models have contributed to our understanding of FPM and FXTAS, and findings from studies using these models are summarized here. This review also discusses how this information is improving our understanding of the molecular and cellular abnormalities that contribute to neurobehavioral features seen in some FPM carriers and in patients with FXTAS. Mouse models show much of the pathology seen in FPM carriers and in individuals with FXTAS, including the presence of elevated levels of Fmr1 mRNA, decreased levels of fragile X mental retardation protein, and ubiquitin-positive intranuclear inclusions. Abnormalities in dendritic spine morphology in several brain regions are associated with neurocognitive deficits in spatial and temporal memory processes, impaired motor performance, and altered anxiety. In vitro studies have identified altered dendritic and synaptic architecture associated with abnormal Ca2+ dynamics and electrical network activity. FPM mice have been particularly useful in understanding the roles of Fmr1 mRNA, fragile X mental retardation protein, and translation of a potentially toxic polyglycine peptide in pathology. Finally, the potential for using these and emerging mouse models for preclinical development of therapies to improve neurological function in FXTAS is considered.

Activation of phagocytic cell NADPH oxidase by norfloxacin: a potential mechanism to explain its bactericidal action

The mechanisms underlying the bactericidal power of fluoroquinolones against intracellular parasites in host macrophages remain poorly understood. We have analyzed the effect of norfloxacin, a fluoroquinolone antibiotic, on the production of reactive oxygen intermediates (O2•− and H2O2) and NADPH oxidase activity in mouse macrophages. The generation of anion superoxide (O2•−) was found to be significantly greater in macrophages incubated with norfloxacin than in untreated controls. This enhancing effect of norfloxacin was dose‐dependent and reached maximal values within 10 min after its addition. The O2•− generated was mainly intracellular, as determined by the use of specific dyes, such as lucigenin and luminol, and able to diffuse freely through the cell membrane. Also, the production of H2O2 was increased in macrophages in response to norfloxacin. The positive effect of norfloxacin was associated to an enhanced mobilization of NADPH oxidase subunits p47phox and p67phox from the cytosol to the plasma membrane in phagocytic cells. The effect of the antibiotic persisted in vivo for several hours. These data support the notion that norfloxacin inhibits mycobacterial growth within phagocytic cells by enhancing intracellular production of O2•− and other reactive oxygen species.

Transcription of liver X receptor is down-regulated by 15-deoxy-Δ12,14-prostaglandin J2 through oxidative stress in human neutrophils

Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.

Human neutrophils synthesize IL-8 in an IgE-mediated activation

It has been demonstrated that neutrophils are responsible for the release of large amounts of the inflammatory chemokine interleukin‐8 (IL‐8), associated with inflammation. To further define the mechanisms implicated, we have analyzed the response of human neutrophils from allergic patients to specific antigens or challenge with anti‐immunoglobulin (Ig)E antibodies. Neutrophils showed a dose‐ and time‐dependent production of IL‐8. The release of the cytokine was parallel to expression of IL‐8 mRNA analyzed by the polymerase chain reaction. This expression was transient—it occurred after 3 h of anti‐IgE treatment and was maintained for 18 h. Trifluoperazine, EGTA, reduced nicotinamide adenine dinucleotide phosphate‐oxidase inhibitors, and reactive oxygen species (ROS) scavengers inhibited IL‐8 production, indicating a critical dependence of calcium and oxidative stress. Moreover, an inhibitory effect of cyclosporin A, an immunosuppressor that inhibits calcineurin activity, on IL‐8 release and IL‐8 mRNA expression was observed. This is the first evidence of the involvement of ROS and calcium/calcineurin in IgE‐dependent IL‐8 production. These findings open new perspectives into the functional role of neutrophils in IgE‐associated diseases.

Heme oxygenase-1 expression is down-regulated by angiotensin II and under hypertension in human neutrophils

Angiotensin II (Ang II) is a peptide hormone able to elicit a strong production of reactive oxygen species by human neutrophils. In this work, we have addressed whether expression of heme oxygenase‐1 (HO‐1), an antioxidant enzyme, becomes altered in these cells upon Ang II treatment or under hypertension conditions. In neutrophils from healthy and hypertensive subjects, induction of HO‐1 mRNA and protein expression with a parallel increase in enzyme activity took place upon treatment with 15‐deoxy‐Δ12,14‐PGJ2 (15dPGJ2). However, Ang II prevented HO‐1 synthesis by normal neutrophils in vitro, and HO‐1 expression was depressed in neutrophils from hypertensive patients in comparison with cells from healthy subjects. In addition, Ang II treatment led to a reduced HO‐1 enzyme activity to levels similar to those found in neutrophils from hypertensive patients. NO donors reversed the inhibition of 15dPGJ2‐dependent HO‐1 expression in neutrophils from hypertensive patients, and conversely, inhibition of inducible NO synthase (NOS2) activity counteracted the stimulatory effect of 15dPGJ2 on HO‐1 expression in normal human neutrophils. Moreover, Ang II canceled 15dPGJ2‐dependent induction of NOS2 mRNA synthesis. Present findings indicate that down‐regulation of HO‐1 expression in neutrophils from hypertensive subjects is likely exerted through the inhibition of NOS2 expression. Additionally, they underscore the potential usefulness of NO donors as new, therapeutic agents against hypertension.

Skewed X Inactivation of the Normal Allele in Fully Mutated Female Carriers Determines the Levels of FMRP in Blood and the Fragile X Phenotype

AbstractBackground: The variable phenotype in female carriers of a full mutation is explained in part by non-random X-chromosome inactivation. The molecular diagnosis of fragile X syndrome is based on the resolution of the number of CGG triplet repeats and the methylation status of a critical CpG in the fragile X mental retardation gene (FMR1) promoter. Neighboring CpGs in the FMR1 promoter are supposed to be equally methylated or unmethylated. Method: Southern blot analysis was performed with double digestion, either with EcoRI/EagI or with HindIII/ SacII. The EagI restriction site was studied by sequencing. The fragile X encoded protein (FMRP) was detected in white blood cells by Western blot. The fragile X phenotype was evaluated by specific clinical examinations. Results: Within one family we found three female carriers of a full mutation and a different degree of methylation of the normal allele that correlated with the levels of FMRP in blood and the fragile X phenotype. Complete methylation at the EagI CpG target (but partially methylated SacII CpG site) was associated with extremely skewed X inactivation (confirmed by analysis of the methylation status at the PGK locus), undetectable FMRP in blood, and a male-like phenotype. Conclusions: In fully mutated female carriers the methylation status at the EagI restriction site correlates with the levels of FMRP in blood and the fragile X phenotype. Neighboring CpG sequences in the FMR1 promoter can be differentially methylated, which should be taken into consideration for molecular diagnosis.

Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes

We have studied the effects of thimerosal, a mercurial compound extensively used as a preservative, as well as other sulfhydryl reagents (e.g. p-hydroxymercurybenzoate, hydrogen peroxide, bromophenacyl bromide, and mercuric chloride) on Ca2+ homeostasis and the redox status of sulfhydryl groups in thymus lymphocytes. They all induced an increase in [Ca2+]i which was blocked with dithiothreitol, suggesting that they act via the oxidation or blockade of sulfhydryl groups. [Ca2+]i increase could be directly related to the effect of the different reagents on cellular protein sulfhydryl content. Experiments with ethidium bromide indicate that the observed rise in [Ca2+]i was not due to a non-specific increase in membrane permeability. Thimerosal differs from the other agents studied in its oxidative properties, which is probably linked to the production of a potent reductor molecule, thiosalicylic acid, which may modulate its oxidative capacity.

A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression

This report focuses on the modulatory role of endogenous H2O2 on lipopolysaccharide (LPS)/interferon‐γ (IFN‐γ)‐induced inducible nitric oxide synthase (NOS2) gene expression in rat peritoneal macrophages. Exogenously added H2O2 was initially found to inhibit the synthesis of NOS2, which prompted us to assess the effect of the activity of monoamine oxidase (MAO) and semicarbazide‐sensitive amine oxidase (SSAO) as H2O2‐forming enzymes on NOS2 gene expression. In the presence of their substrates, tyramine for MAO and benzylamine for SSAO, intracellular synthesis of H2O2 took place with concomitant inhibition of LPS/IFN‐γ‐induced NOS2 protein synthesis, as detected by Western blotting, flow cytometry, and immunofluorescence microscopy analyses. Pargyline and semicarbazide, specific inhibitors of MAO and SSAO, respectively, canceled this negative effect of MAO substrates on NOS2 expression. In the presence of Fe2+ and Cu2+ ions, inhibition of NOS2 expression was enhanced, suggesting the participation in this regulation of species derived from Fenton chemistry. In addition, the negative effect of H2O2, generated by MAOs, was found to be exerted on NOS2 mRNA levels. These data offer a new insight in the control of NOS2 expression through the intracellular levels of H2O2 and other reactive oxygen species (ROS). The hypothesis can be raised that the inhibition of NOS by H2O2 could constitute a protective mechanism against the cytotoxic consequences of the activation of ROS‐generating enzymes, thus providing a new, singular role for the MAO family of proteins.