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1.
Article in English | MEDLINE | ID: mdl-38445953

ABSTRACT

RATIONALE: Nitric oxide (NO) is elevated in the airways and serum of allergic asthmatic patients, suggesting an important role in asthma. NO production has been widely attributed to the canonical inducible nitric oxide synthase (iNOS). Much effort has been made to inhibit this enzyme with two outcomes: no asthma improvement; and partial NO reduction, suggesting the involvement of an iNOS-independent source. OBJECTIVES: Neutrophils produce NO under inflammatory conditions and their role in asthma has been overlooked. The present study analyzes their possible role as source of NO. METHODS: Our hypothesis was tested in 99 allergic patients with intermittent bronchial asthma and 26 healthy donors. NO production by blood and sputum neutrophils in response to allergens, anti-IgE, and anti-IgE receptors Abs was assessed by Griess, flow cytometry and confocal microscopy. Extracellular traps (ETs) formation, as a possible consequence of NO production, was quantified by western blot and confocal microscopy, and reactive oxygen species by luminol-enhanced chemiluminescence. RESULTS: Among blood and sputum granulocytes from allergic asthmatic patients, only neutrophils, produce NO by an IgE-dependent mechanism. This production is independent of NOS, but dependent on a reaction between L-arginine and reactive oxygen species from NOX2. NO and ETosis are induced in parallel, and NO amplifies ETs formation, which is a key mediator in asthma. CONCLUSIONS: Our findings reveal a novel role of neutrophils as the unique allergen/IgE-dependent NO source in allergic asthma enhancing ETs formation. These results suggest that NO produced by neutrophils needs further consideration in the treatment of allergic asthma.

2.
Front Immunol ; 13: 1015529, 2022.
Article in English | MEDLINE | ID: mdl-36518751

ABSTRACT

Background: Neutrophils are involved in the pathophysiology of allergic asthma, where the Eosinophil Cationic Protein (ECP) is a critical inflammatory mediator. Although ECP production is attributed to eosinophils, we reported that ECP is also present in neutrophils from allergic patients where, in contrast to eosinophils, it is produced in an IgE-dependent manner. Given the key role of ECP in asthma, we investigated the molecular mechanisms involved in ECP production as well as the effects induced by agonists and widely used clinical approaches. We also analyzed the correlation between ECP production and lung function. Methods: Neutrophils from allergic asthmatic patients were challenged with allergens, alone or in combination with cytokines, in the presence of cell-signaling inhibitors and clinical drugs. We analyzed ECP levels by ELISA and confocal microscopy. Lung function was assessed by spirometry. Results: IgE-mediated ECP release is dependent on phosphoinositide 3-kinase, the extracellular signal-regulated kinase (ERK1/2) and the production of reactive oxygen species by NADPH-oxidase. Calcineurin phosphatase and the transcription factor NFAT are also involved. ECP release is enhanced by the cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor, and inhibited by interferon-γ, IL-10, clinical drugs (formoterol, tiotropium and budesonide) and allergen-specific IT. We also found an inverse correlation between asthma severity and ECP levels. Conclusions: Our results suggest the molecular pathways involved in ECP production and potential therapeutic targets. We also provide a new method to evaluate disease severity in asthmatic patients based on the quantification of in vitro ECP production by peripheral neutrophils.


Subject(s)
Asthma , Hypersensitivity , Humans , Eosinophil Cationic Protein/metabolism , Neutrophils/metabolism , Phosphatidylinositol 3-Kinases , Allergens , Asthma/drug therapy , Asthma/metabolism , Cytokines/metabolism , Immunoglobulin E
3.
FASEB J ; 35(5): e21483, 2021 05.
Article in English | MEDLINE | ID: mdl-33788304

ABSTRACT

Histamine is a critical inflammatory mediator in allergic diseases. We showed in a previous work that neutrophils from allergic patients produce histamine in response to allergens to which the patients were sensitized. Here, we investigate the molecular mechanisms involved in this process using peripheral blood neutrophils. We challenged these cells in vitro with allergens and analyzed histamine release in the culture supernatants. We also explored the effect of common therapeutic drugs that ameliorate allergic symptoms, as well as allergen-specific immunotherapy. Additionally, we examined the expression of histidine decarboxylase and diamine oxidase, critical enzymes in the metabolism of histamine, under allergen challenge. We show that allergen-induced histamine release is dependent on the activation of the phosphoinositide 3-kinase, mitogen-activated protein kinase p38, and extracellular signal-regulated kinase 1/2 signaling pathways. We also found a contribution of the phosphatase calcineurin to lesser extent. Anti-histamines, glucocorticoids, anti-M3-muscarinic receptor antagonists, and mainly ß2 -receptor agonists abolished the allergen-dependent histamine release. Interestingly, allergen-specific immunotherapy canceled the histamine release through the downregulation of histidine decarboxylase expression. Our observations describe novel molecular mechanisms involved in the allergen-dependent histamine release by human neutrophils and provide new targets to inhibit histamine production.


Subject(s)
Allergens/adverse effects , Asthma/drug therapy , Histamine Release/drug effects , Histamine/metabolism , Hypersensitivity/drug therapy , Immunotherapy/methods , Neutrophils/immunology , Asthma/etiology , Asthma/pathology , Case-Control Studies , Humans , Hypersensitivity/etiology , Hypersensitivity/pathology , Neutrophils/drug effects
4.
PLoS One ; 15(2): e0227897, 2020.
Article in English | MEDLINE | ID: mdl-32023281

ABSTRACT

The optic lobes of the fruit fly Drosophila melanogaster form a highly wired neural network composed of roughly 130.000 neurons of more than 80 different types. How neuronal diversity arises from very few cell progenitors is a central question in developmental neurobiology. We use the optic lobe of the fruit fly as a paradigm to understand how neuroblasts, the neural stem cells, generate multiple neuron types. Although the development of the fly brain has been the subject of extensive research, very little is known about the lineage relationships of the cell types forming the adult optic lobes. Here we perform a large-scale lineage bioinformatics analysis using the graph theory. We generated a large collection of cell clones that genetically label the progeny of neuroblasts and built a database to draw graphs showing the lineage relationships between cell types. By establishing biological criteria that measures the strength of the neuronal relationships and applying community detection tools we have identified eight clusters of neurons. Each cluster contains different cell types that we pose are the product of eight distinct classes of neuroblasts. Three of these clusters match the available lineage data, supporting the predictive value of the analysis. Finally, we show that the neuronal progeny of a neuroblast do not have preferential innervation patterns, but instead become part of different layers and neuropils. Here we establish a new methodology that helps understanding the logic of Drosophila brain development and can be applied to the more complex vertebrate brains.


Subject(s)
Cell Lineage , Drosophila melanogaster/cytology , Neurons/cytology , Optic Lobe, Nonmammalian/cytology , Animals , Clone Cells , Reproducibility of Results
5.
Cell ; 173(2): 485-498.e11, 2018 04 05.
Article in English | MEDLINE | ID: mdl-29576455

ABSTRACT

Understanding how complex brain wiring is produced during development is a daunting challenge. In Drosophila, information from 800 retinal ommatidia is processed in distinct brain neuropiles, each subdivided into 800 matching retinotopic columns. The lobula plate comprises four T4 and four T5 neuronal subtypes. T4 neurons respond to bright edge motion, whereas T5 neurons respond to dark edge motion. Each is tuned to motion in one of the four cardinal directions, effectively establishing eight concurrent retinotopic maps to support wide-field motion. We discovered a mode of neurogenesis where two sequential Notch-dependent divisions of either a horizontal or a vertical progenitor produce matching sets of two T4 and two T5 neurons retinotopically coincident with pairwise opposite direction selectivity. We show that retinotopy is an emergent characteristic of this neurogenic program and derives directly from neuronal birth order. Our work illustrates how simple developmental rules can implement complex neural organization.


Subject(s)
Drosophila/physiology , Motion Perception/physiology , Retina/physiology , Animals , Drosophila Proteins/metabolism , Locomotion/physiology , Models, Neurological , Neurons/physiology , Optic Lobe, Nonmammalian/chemistry , Optic Lobe, Nonmammalian/metabolism , Receptors, Notch/metabolism , Retina/cytology , Visual Pathways
6.
Nature ; 541(7637): 365-370, 2017 01 19.
Article in English | MEDLINE | ID: mdl-28077877

ABSTRACT

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. Here we show that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors. Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy.


Subject(s)
Body Patterning , Cell Differentiation , Drosophila melanogaster/cytology , Neurogenesis , Neurons/cytology , Optic Lobe, Nonmammalian/cytology , Animals , Body Patterning/genetics , Brain/cytology , Brain/growth & development , Brain/metabolism , Cell Movement , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Female , Male , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/metabolism , Neuropil/cytology , Neuropil/metabolism , Optic Lobe, Nonmammalian/growth & development , Optic Lobe, Nonmammalian/metabolism , Pupa/cytology , Pupa/genetics , Pupa/growth & development , Spatio-Temporal Analysis , Time Factors
7.
Cell Rep ; 15(4): 774-786, 2016 Apr 26.
Article in English | MEDLINE | ID: mdl-27149843

ABSTRACT

How neuronal and glial fates are specified from neural precursor cells is an important question for developmental neurobiologists. We address this question in the Drosophila optic lobe, composed of the lamina, medulla, and lobula complex. We show that two gliogenic regions posterior to the prospective lamina also produce lamina wide-field (Lawf) neurons, which share common progenitors with lamina glia. These progenitors express neither canonical neuroblast nor lamina precursor cell markers. They bifurcate into two sub-lineages in response to Notch signaling, generating lamina glia or Lawf neurons, respectively. The newly born glia and Lawfs then migrate tangentially over substantial distances to reach their target tissue. Thus, Lawf neurogenesis, which includes a common origin with glia, as well as neuronal migration, resembles several aspects of vertebrate neurogenesis.

8.
Nat Methods ; 9(1): 47-55, 2011 Dec 28.
Article in English | MEDLINE | ID: mdl-22205518

ABSTRACT

The development of two-component expression systems in Drosophila melanogaster, one of the most powerful genetic models, has allowed the precise manipulation of gene function in specific cell populations. These expression systems, in combination with site-specific recombination approaches, have also led to the development of new methods for clonal lineage analysis. We present a hands-on user guide to the techniques and approaches that have greatly increased resolution of genetic analysis in the fly, with a special focus on their application for lineage analysis. Our intention is to provide guidance and suggestions regarding which genetic tools are most suitable for addressing different developmental questions.


Subject(s)
Drosophila melanogaster/genetics , Gene Expression , Animals , Bacterial Proteins/genetics , Cell Lineage , Clonal Evolution , Clone Cells/physiology , Drosophila Proteins/physiology , Genetic Techniques , Serine Endopeptidases/genetics , Transcription Factors/physiology , Transgenes/genetics
9.
Circ Res ; 108(11): 1348-57, 2011 May 27.
Article in English | MEDLINE | ID: mdl-21493898

ABSTRACT

BACKGROUND: Sustained vascular smooth muscle contraction is mediated by extracellular Ca(2+) influx through L-type voltage-gated Ca(2+) channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca(2+) sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca(2+) release from the sarcoplasmic reticulum (calcium channel-induced Ca(2+) release). We have studied the functional role of calcium channel-induced Ca(2+) release and the inter-relations between Ca(2+) channel and RhoA/ROCK activation. METHODS AND RESULTS: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca(2+) and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca(2+) channel activation and subsequent metabotropic Ca(2+) release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca(2+) from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca(2+) release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca(2+) influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. CONCLUSIONS: These findings reveal that calcium channel-induced Ca(2+) release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca(2+) channel activation with metabotropic Ca(2+) release and sensitization (RhoA/ROCK stimulation).


Subject(s)
Calcium Channels, L-Type/physiology , Muscle, Smooth, Vascular/physiology , Vasoconstriction/physiology , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism , Animals , Basilar Artery/physiology , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/genetics , Coronary Vasospasm/physiopathology , Electric Stimulation , Enzyme Inhibitors/pharmacology , Hypertension/physiopathology , Indoles/pharmacology , Mice , Mice, Knockout , Muscle Contraction/drug effects , Muscle Contraction/physiology , Nifedipine/pharmacology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Rabbits , Rats , Sarcoplasmic Reticulum/physiology , Vasoconstriction/drug effects , Vasospasm, Intracranial/physiopathology , rho GTP-Binding Proteins/antagonists & inhibitors , rho-Associated Kinases/antagonists & inhibitors , rhoA GTP-Binding Protein
10.
Nat Methods ; 6(8): 600-2, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19633664

ABSTRACT

In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.


Subject(s)
Cell Lineage , Drosophila melanogaster/genetics , Genomics/methods , Mitosis , Recombination, Genetic , Animals , Clone Cells , Drosophila melanogaster/cytology , Fluorometry , Genomics/instrumentation , Mutation
11.
Cell Calcium ; 42(4-5): 513-20, 2007.
Article in English | MEDLINE | ID: mdl-17559931

ABSTRACT

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.


Subject(s)
Calcium Channels, L-Type/physiology , Calcium/metabolism , Muscle, Smooth, Vascular/metabolism , Animals , Arteries/metabolism , Arteries/physiology , Calcium Signaling , Inositol 1,4,5-Trisphosphate/metabolism , Models, Biological , Muscle Contraction , Muscle, Smooth, Vascular/physiology , Rats , Type C Phospholipases/metabolism
12.
Proc Natl Acad Sci U S A ; 103(11): 4316-21, 2006 Mar 14.
Article in English | MEDLINE | ID: mdl-16537528

ABSTRACT

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium Channels, L-Type/metabolism , Calcium Signaling/physiology , Myocytes, Smooth Muscle/physiology , Adenosine Triphosphate/pharmacology , Animals , Basilar Artery/cytology , Basilar Artery/drug effects , Basilar Artery/physiology , Calcium Channels, L-Type/drug effects , Calcium Signaling/drug effects , In Vitro Techniques , Muscle Contraction/drug effects , Muscle Contraction/physiology , Myocytes, Smooth Muscle/drug effects , Patch-Clamp Techniques , Rats , Rats, Wistar , Vasoconstriction/drug effects , Vasoconstriction/physiology
13.
EMBO J ; 22(17): 4337-45, 2003 Sep 01.
Article in English | MEDLINE | ID: mdl-12941686

ABSTRACT

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca2+] owing to either Ca2+ influx through voltage-gated Ca2+ channels of the plasmalemma or receptor-mediated Ca2+ release from the sarcoplasmic reticulum (SR). We show that voltage-gated Ca2+ channels in arterial myocytes mediate fast Ca2+ release from the SR and contraction without the need of Ca2+ influx. After sensing membrane depolarization, Ca2+ channels activate G proteins and the phospholipase C-inositol 1,4,5-trisphosphate (InsP3) pathway. Ca2+ released through InsP3-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca2+ signal. These observations demonstrate a new mechanism of signaling SR Ca(2+)-release channels and reveal an unexpected function of voltage-gated Ca2+ channels in arterial myocytes. Our findings may have therapeutic implications as the calcium-channel-induced Ca2+ release from the SR can be suppressed by Ca(2+)-channel antagonists.


Subject(s)
Calcium Channels/metabolism , Myocytes, Smooth Muscle/physiology , Sarcoplasmic Reticulum/metabolism , Animals , Calcium Signaling , GTP-Binding Proteins/metabolism , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/metabolism , Membrane Potentials , Muscle Contraction/physiology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Rats , Rats, Wistar , Ryanodine Receptor Calcium Release Channel/metabolism , Type C Phospholipases/metabolism
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