Antonio Vega

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.

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.

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.

Allergen immunotherapy decreases LPS-induced NF-κB activation in neutrophils from allergic patients

Allergen‐specific immunotherapy (IT) is widely used to treat allergic diseases. The molecular mechanisms have not been clarified yet completely. The present work was undertaken to analyze the effect of IT in the activation of NF‐κB.

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.