Regulation of Neutrophil Degranulation and Cytokine Secretion: A Novel Model Approach Based on Linear Fitting

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes

Regulation paracrine de la contractilite du muscle lisse bronchique

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Rôle de la mobilisation calcique dans la régulation de la réponse oxydative dans un modèle de neutrophiles humains

En réponse à un stimulus, la réponse oxydante suite à l'assemblage de la NADPH oxydase est une fonction biologique fondamentale des phagocytes. Une réponse exagérée se caractérise par la pérennisation de l'inflammation. Ce travail a pour but de caractériser la relation entre l'élévation de la concentration de Ca2+ libre cytosolique ([Ca2+]i) et la réponse oxydante, induite par le N-formyl-L-Méthionyl-L-Leucyl-L-Phénylalanine (fMLF), dans un modèle de neutrophiles humains : les cellules HL-60 différenciées. La mesure simultanée de la [Ca2+]i et de la production d'H2O2 nous a permis de montrer que l'entrée de Ca2+ extracellulaire, activée par la vidange des stocks intracellulaires de Ca2+, est nécessaire à l'initiation de la réponse biologique en réponse au fMLF. L'ADP-ribose cyclique est impliquée dans cette mobilisation calcique. En outre, la sensibilisation des cellules par l'interleukine-8 augmente la production d'H2O2 induite par le fMLF et est dépendante d'une entrée de Ca2+ extracellulaire. Par une approche biochimique nous avons établi que la translocation membranaire des protéines Rac (nécessaires à l'activation de la NADPH oxydase) est dépendante de l'élévation de la [Ca2+]i, tandis que par une approche moléculaire nous avons identifié les canaux potentiellement impliqués dans l'entrée capacitive de Ca2+. La réponse oxydante, via la translocation de Rac, est dépendante d'une entrée de Ca2+ extracellulaire. Elle répond au mécanisme d'entrée capacitive résultant de l'activation de canaux calciques (récepteurs potentiels transitoires) par différents messagers mobilisateurs du Ca2+.In response to a stimulus, the oxidative response, resulting of the NADPH oxidase assembly, is a fundamental biological function of phagocytic cells. An excessive response is associated to chronical inflammations. In this study, we have investigated the relation between the elevation of cytosolic-free calcium concentration ([Ca2+]i) and oxydative response, induced by N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLF), in a model of neutrophils: differentiated HL-60 cells. We have demonstrated, by simultaneously measurements of [Ca2+]i and H2O2 production, that extracellular Ca2+ entry, regulated by the intracellular Ca2+ release stores, is necessary to the biological response initiation in response to fMLF. Cyclic ADP-ribose is implicated in this calcium mobilization. Furthermore, interleukin-8 priming of cells increases H2O2 production dependently of extracellular Ca2+ entry. By biochemical studies, we have estasblished that the translocation of Rac proteins is tightly correlated to an [Ca2+]i elevation, whereas with molecular studies we have identified capacitative channels probably implicated in the extracellular Ca2+ entry. The oxydative response, via Rac translocation is dependent on extracellular Ca2+ entry by the capacitative mechanism resulting of the calcium channels activation (potentiel transient receptors) stimulated by differents second messengers mobilizing Ca2+.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Human umbilical cord blood monocyte-derived eosinophils produce superoxide but not nitric oxide

Both N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine, and 1-O-Alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine induced superoxide release in umbilical cord eosinophils, while no response was observed with lipopolysaccharide, interleukin-4 and/or interferon-gamma. Furthermore, upon activation with different inflammatory stimuli, neither induction of nitric oxide synthesis nor expression of the constitutive and/or inducible nitric oxide synthase were observed in these eosinophils derived in vitro. Human umbilical cord derived eosinophils are able to produce superoxide as peripheral blood eosinophils. Whether human peripheral eosinophils are capable of NO synthesis is still the subject of considerable debate, nevertheless, our results suggest that these in vitro derived eosinophils are not capable of nitric oxide synthesis

Selective differentiation and proliferation of human eosinophils from umbilical cord blood: calcium fluxes and superoxide ion secretion

Investigation of the physiologic mechanisms involved in the activation of eosinophils is crucial to comprehend their role in the pathogenesis of allergic reactions. To overcome the difficulty of obtaining large numbers of eosinophils, we differentiated in vitro eosinophils from human umbilical cord blood mononuclear cells. These cells responded to fMLP or PAF with an increase in [Ca2+]i, associated with O2 production. Deprivation or chelation of extracellular calcium induced a reduction of fMLP or PAF-induced [Ca2+]i rise and O2- production. Similar results were obtained with extracellular Ni2+ addition. Chelation of intracellular calcium induced an inhibition of fMLP- or PAF-induced [Ca2+]i rise and a decrease in O2- production. Our results indicate that fMLP- and PAF-dependent O2- production in eosinophils requires intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation

Vieillissement artériel et sensibilité au calcium de la contraction (couplage entre récepteurs [alpha]1-adrénergiques et protéines G[indice i/o])

NANCY1-SCD Pharmacie-Odontologie (543952101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Nedocromil sodium inhibits IgE- and IgG-related antigen-induced contraction in guinea-pig trachea

The effects of nedrocromil sodium and disodium cromoglycate were studied on the anaphylactic contraction of guinea-pig trachea in two models of active sensitization (IgE and IgG models). The influence of epithelial removal on the effects of nedocromil sodium and disodium cromoglycate was examined because several studies have shown that the epithelial layer can modulate agonist- or antigen-induced contractile responses. Disodium cromoglycate (10(-4) M) and nedocromil sodium (10(-4) M) provided significant protection against antigen-induced contractions of guinea-pig tracheal smooth muscle in the IgG model. But only nedocromil sodium had an effect at this concentration in the IgG model and was also effective at 10(-5) M in the epithelium-denuded tracheal strips. At this concentration, disodium cromoglycate lost its protective effect. Comparison with the results obtained with FPL-55712, AA-861 and mepyramine suggested that these drugs affect histamine and particularly leukotriene synthesis and/or release by mast cells or other immunocompetent cells. These findings indicate that nedocromil sodium inhibits the IgE- and IgG-related antigen-induced contraction in guinea-pig airways, whereas disodium cromoglycate inhibits only the IgG-related processes. This study supports the hypothesis that these drugs modulate antigen-induced mediator synthesis and/or release from immunocompetent cells