Gonzalo Alba

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.

Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up‐regulation of its COX‐2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX‐2 expression becomes highly induced by anti‐immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE2 and thromboxane A2 release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4‐(2‐aminoethyl)benzenesulphonyl fluoride and 4‐hydroxy‐3‐methoxyaceto‐phenone, completely cancelled anti‐IgE‐induced COX‐2 protein up‐regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen‐activated protein kinases (MAPKs), p38 and extracellular signal‐regulated kinase, and also, the transcription factor, nuclear factor (NF)‐κB, are involved in the up‐regulation of COX‐2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF‐κB pathway, such as N(α)‐benzyloxycarbonyl‐l‐leucyl‐l‐leucyl‐l‐leucinal, abolished IgE‐dependent COX‐2 induction. Evidence is also presented, using Fe2+/Cu2+ ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti‐IgE‐elicited COX‐2 expression through MAPK and NF‐κB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX‐2 expression by human neutrophils in allergic conditions.

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.

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcϵ-receptor antibodies. Antigens, anti-IgE and anti-FcϵRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE2) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

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.

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.

Induction of cyclooxygenase-2 expression by allergens in lymphocytes from allergic patients.

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up‐regulation of COX‐2 expression is responsible for increased PG release during inflammatory conditions and is thought to be also involved in allergic states. In this study, we demonstrate that in human T, B and natural killer lymphocytes from allergic patients, COX‐2 expression became induced upon cell challenge with specific allergens and that this process is presumably IgE dependent and occurs after CD23 receptor ligation. This induction took place at both mRNA and protein levels and was accompanied by PGD2 release. IgE‐dependent lymphocyte treatment elicited, in parallel, an activation of the MAPK p38 and extracellular signal‐regulated kinase 1/2, an enhancement of calcineurin (CaN) activity, and an increase of the DNA‐binding activity of the nuclear factor of activated T cells and of NF‐κB, with a concomitant decrease in the levels of the cytosolic inhibitor of κB, IκB. In addition, specific chemical inhibitors of MAPK, such as PD098059 and SB203580, as well as MG‐132, an inhibitor of proteasomal activity, abolished allergen‐induced COX‐2 up‐regulation, suggesting that this process is mediated by MAPK and NF‐κB. However, induction of COX‐2 expression was not hampered by the CaN inhibitor cyclosporin A. We also examined the effect of a selective COX‐2 inhibitor, NS‐398, on cytokine production by human lymphocytes. Treatment with NS‐398 severely diminished the IgE‐dependently induced production of IL‐8 and TNF‐α. These results underscore the relevant role of lymphocyte COX‐2 in allergy and suggest that COX‐2 inhibitors may contribute to the improvement of allergic inflammation through the reduction of inflammatory mediator production by human lymphocytes.

Platelet-activating factor downregulates the expression of liver X receptor-α and its target genes in human neutrophils

Liver X receptors (LXRs) are ligand‐activated members of the nuclear receptor superfamily that regulate the expression of genes involved in lipid metabolism and inflammation, although their role in inflammation and immunity is less well known. It has been reported that oxysterols/LXRs may act as anti‐inflammatory molecules, although opposite actions have also been reported. In this study, we investigated the effect of platelet‐activating factor (PAF), a proinflammatory molecule, on LXRα signalling in human neutrophils. We found that PAF exerted an inhibitory effect on mRNA expression of TO901317‐induced LXRα, ATP‐binding cassette transporter A1, ATP‐binding cassette transporter G1, and sterol response element binding protein 1c. This negative action was mediated by the PAF receptor, and was dependent on the release of reactive oxygen species elicited by PAF, as it was enhanced by pro‐oxidant treatment and reversed by antioxidants. Current data also support the idea that PAF induces phosphorylation of the LXRα molecule in an extracellular signal‐regulated kinase 1/2‐mediated fashion. These results suggest that a possible mechanism by which PAF exerts its proinflammatory effect is through the downregulation of LXRα and its related genes, which supports the notion that LXRα ligands exert a modulatory role in the neutrophil‐mediated inflammatory response.

Calcineurin expression and activity is regulated by the intracellular redox status and under hypertension in human neutrophils

Calcineurin (protein phosphatase 2B) (CN) comprises a family of serine/threonine phosphatases that play a pivotal role in signal transduction cascades in a variety of cells, including neutrophils. Angiotensin II (Ang II) increases both activity and de novo synthesis of CN in human neutrophils. This study focuses on the role that intracellular redox status plays in the induction of CN activity by Ang II. Both de novo synthesis of CN and activity increase promoted by Ang II were downregulated when cells were treated with L-buthionine-(S,R)-sulfoximine, an inhibitor of synthesis of the antioxidant glutathione. We have also investigated the effect of pyrrolidine dithiocarbamate and phenazine methosulfate, which are antioxidant and oxidant compounds, respectively, and concluded that the intracellular redox status of neutrophils is highly critical for Ang II-induced increase of CN expression and activity. Results obtained in neutrophils from hypertensive patients were very similar to those obtained in these cells on treatment with Ang II. We have also addressed the possible functional implication of CN activation in the development of hypertension. Present findings indicate that downregulation of hemoxygenase-1 expression in neutrophils from hypertensive subjects is likely mediated by CN, which acts by hindering translocation to the nucleus of the transcription factor NRF2. These data support and extend our previous results and those from other authors on modulation of CN expression and activity levels by the intracellular redox status.