Pedro Chacón

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.

Neutrophils as a Novel Source of Eosinophil Cationic Protein in IgE-Mediated Processes

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with 35S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcεRI and galectin-3 (FcεRI>galectin-3), but not against FcεRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

Amyloid β serves as an NGF-like neurotrophic factor or acts as a NGF antagonist depending on its concentration

In the nervous system, both the shape and connectivity of neurons are strongly influenced by soluble, extracellular factors. Indeed, we recently demonstrated that after binding to p75NTR, the common neurotrophin receptor, nerve growth factor (NGF) controls the morphology and connectivity of cultured mouse hippocampal neurons by encouraging the production of fewer yet longer dendrites, and by augmenting GABAergic connectivity. These effects of NGF are mediated by the differential expression of Enhancer‐of‐split 1/5 homologs and neurogenin 3. Amyloid β (Aβ), a pathogenic agent in Alzheimer’s disease (AD) is known to bind to p75NTR, hence we studied its influence on cultured hippocampal neurons. At 800 nM, Aβ(1–40) prevents NGF‐induced activation of NF‐κB and consequently, it depresses the expression of Enhancer‐of‐split 1. Thus, at this concentration, the effect of Aβ on neurons is antagonistic to those provoked by NGF and accordingly, neurons sprout more yet shorter dendrites and their GABAergic input decreases. In contrast, at lower concentration, 20 nM, the amyloid induces cellular effects similar to those induced by NGF, both in terms of gene expression, neuronal morphology, and GABAergic connectivity. Our results demonstrate that Aβ may act as a neurotrophic factor that mimics the activity of NGF. However, at higher concentrations, the amyloid behaves as an antagonist of NGF, contributing to the advent of AD.

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.

Specific Allergens Enhance Elastase Release in Stimulated Neutrophils from Asthmatic Patients

Background: The presence of the three forms of IgE receptor – the heterotrimeric high-affinity receptor for IgE (FcΕRI), the low-affinity receptor for IgE (FcΕRII/CD23) and the Mac-2/IgE-binding protein (ΕBP) – has been demonstrated on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present at the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes, such as elastase, that might potentiate inflammation. It is not known whether specific allergens are able to elicit elastase release by neutrophils from allergic patients. In addition, we attempted to evaluate the relationship between neutrophil degranulation and lung function of the patients, measured as FEV1. Methods: Neutrophils were challenged in vitro with the specific allergens that produced clinical symptoms in asthmatic patients. The cells were also challenged with allergen to which the patients were not sensitive. Neutrophils from normal subjects were challenged with allergens as control. Results: The in vitro challenge of neutrophils with allergens to which the patients were sensitive elicited a release of elastase by these cells. The in vitro activation of neutrophils was highly allergen specific; allergens other than those accounting for clinical symptoms did not evoke elastase release, and allergens were ineffective on neutrophils from healthy donors. A significant inverse correlation was observed between elastase release and patients’ lung function, measured as FEV1. Conclusion: An IgE-dependent mechanism might promote elastase release by neutrophils at allergic sites. There is a significant inverse relationship between levels of elastase released by neutrophils from allergic patients and lung function, as assessed by FEV1.

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.

Histamine production by human neutrophils

Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine‐producing cells. Neutrophils store ~0.29 pg/cell and release ~50% of the histamine content in an antigen‐dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate‐limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001‐0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.—Alcañiz, L., Vega, A., Chacón, P., El Bekay, R., Ventura, I., Aroca, R., Blanca, M., Bergstralh, D. T., Monteseirin, J. Histamine production by human neutrophils. FASEB J. 27, 2902–2910 (2013). www.fasebj.org

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.

NGF-activated protein tyrosine phosphatase 1B mediates the phosphorylation and degradation of I-κ-Bα coupled to NF-κ-B activation, thereby controlling dendrite morphology

NGF diminishes dendrite complexity in cultured hippocampal neurons by decreasing the number of primary and secondary dendrites, while increasing the length of those that remain. The transduction pathway used by NGF to provoke dendrite elongation involves the activation of NF-kappa-B and the expression of the homologues of Enhancer-of-split 1 gene. Here, we define important steps that link NGF with NF-kappa-B activation, through the activity of protein tyrosine phosphatase 1B (PTP1B). Binding of NGF to p75(NTR) stimulates PTP1B activity, which can be blocked by either pharmacological inhibition of the phosphatase or by transfecting neurons with a dn PTP1B isoform, whereby NGF is no longer able to stimulate dendrite growth. Indeed, overexpressing PTP1B alone provoked dendrite growth and further studies revealed a role for the src kinase downstream of PTP1B. Again, loss of src activity largely cancelled out the capacity of NGF to promote dendrite growth, whereas overexpression of v-src in neurons was sufficient to promote dendrite growth. Finally, the NGF/p75(NTR)/PTP1B/src kinase pathway led to the tyrosine phosphorylation of I-kappa-Balpha prior to its degradation, an event that is necessary for NF-kappa-B activation. Indeed, the dendrite growth response to NGF was lost when neurons were transfected with a mutant form of I-kappa-Balpha that lacks tyr42. Thus, our data suggest that PTP1B fulfils a central role in the NGF signalling that controls dendrite patterning in hippocampal neurons.