miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils.

The release of cytokines by neutrophils constitutes an essential process in the development of inflammation by recruiting and activating additional cells. Neutrophils are also able to secrete a complex of S100A8 and S100A9 proteins (S100A8/A9), which can amplify the general inflammatory state of the host and is involved in the pathogenesis of several chronic inflammatory diseases, such as rheumatoid arthritis (RA). S100A8/A9 have received renewed attention due to their susceptibility to several function-altering post-translational modifications. In that context, it has been recently demonstrated that only the phosphorylated form of S100A8/A9 (S100A8/A9-P) is able to induce the secretion of several cytokines in neutrophils. Here, we investigate the mechanism by which this post-translational modification of S100A8/A9 can regulate the extracellular activity of the protein complex and its impact on the inflammatory functions of neutrophils. We found that S100A8/A9-P are present in large amounts in the synovial fluids from RA patients, highlighting the importance of this form of S100A8/A9 complex in the inflammation process. Using miRNA-sequencing on S100A8/A9-P-stimulated differentiated HL-60 cells, we identified a dysregulation of miR-146a-5p and miR-155-5p expression through TRL4 signaling pathways. Our data reveal that overexpression of these miRNAs in neutrophil-like cells reduces S100A8/A9-P-mediated secretion of pro-inflammatory cytokines

miRNA-132-5p mediates a negative feedback regulation of IL-8 secretion through S100A8/A9 downregulation in neutrophil-like HL-60 cells

BackgroundNeutrophils are an important source of pro-inflammatory and immunomodulatory cytokines. This makes neutrophils efficient drivers of interactions with immune and non-immune cells to maintain homeostasis and modulate the inflammatory process by notably regulating the release of cytokines. Ca2+-dependent regulatory mechanism encompassing cytokine secretion by neutrophils are not still identified. In this context, we propose to define new insights on the role of Ca2+-binding proteins S100A8/A9 and on the regulatory role of miRNA-132-5p, which was identified as a regulator of S100A8/A9 expression, on IL-8 secretion.MethodsDifferentiated HL-60 cells, a human promyelocytic leukemia cell line that can be induced to differentiate into neutrophil-like cells, were used as a model of human neutrophils and treated with N- formyl-methionyl-leucyl-phenylalanine (fMLF), a bacterial peptide that activates neutrophils. shRNA knockdown was used to define the role of selected targets (S100A8/A9 and miRNA-132-5p) on IL-8 secretion.Results and discussionDifferent types of cytokines engage different signaling pathways in the secretion process. IL-8 release is tightly regulated by Ca2+ binding proteins S100A8/A9. miRNA-132-5p is up-regulated over time upon fMLF stimulation and decreases S100A8/A9 expression and IL-8 secretion.ConclusionThese findings reveal a novel regulatory loop involving S100A8/A9 and miRNA-132-5p that modulates IL-8 secretion by neutrophils in inflammatory conditions. This loop could be a potential target for therapeutic intervention in inflammatory diseases.</jats:sec

Activation non-receptorielle des mastocytes par les kinines et les polyamines naturelles

SIGLEINIST T 77534 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Oxatomide and calcium: mechanisms involved in the secretion of mast cell mediators

Human cutaneous mast cells and their experimental model rat peritoneal mast cells, can be stimulated by an IgE-dependent process or by peptides through the direct activation of G proteins. Both activation pathways lead to the increase of cytosolic Ca2+ level. This increase in dependent of the mobilisation of intracellular calcium stores of the endoplasmic reticulum involving the stimulation of IP3-sensitive calcium channels. Mast cells are characterized by the absence of calcium channels in the plasma membrane. Oxatomide has been synthetized as an analog of cinnarizine. However oxatomide is inactive on current calcium channels. In mast cells, oxatomide inhibits the increase of cytosolic calcium elicited during mast cell activation. Consequently mast cell exocytosis is inhibited altogether with the release of newly synthetized mediators. The authors propose several putative targets for oxatomide in mast cells. The therapeutic effect of oxatomide is also related to its property to antagonize the effects of anaphylactic mediators on their selective receptors

Stress oxydatif, inflammation vasculaire et métalloprotéinases (étude in vitro sur un modèle de coculture)

Au cours de l athérosclérose, l invasion de la paroi vasculaire par les leucocytes conduit à l inflammation de ce tissu et à l établissement d un stress oxydatif. Ce travail aborde les effets du stress oxydatif et les effets d interactions potentielles entre un modèle de neutrophiles (?HL60) et des cellules endothéliales (HCAEC) ou musculaires lisses (HCSMC) de coronaires humaines sur les métalloprotéinases (MMPs) cellulaires et la mobilité des ?HL60. Au cours des passages, la lignée promyélocytaire HL60, différenciée en modèle de neutrophiles ?HL60, subit des variations d expression et de production de MMP9 et de ses propriétés de mobilité. Les espèces réactives de l oxygène (ERO) d origine biochimique ou cellulaire ne modifient pas l activité MMP2 ou MMP9 des HCAEC et HCSMC. Il n y a pas modification des activités MMPs en coculture ?HL60/HCAEC mais stimulation de l activité MMP9 en coculture ?HL60/HCSMC. Les HCSMC stimulent les capacités migratoires et favorisent la réponse invasive des ?HL60 au N-formyl-L-Méthionyl-L-Leucyl-L-Phénylalanine. Les enzymes antioxydantes ont peu d effet sur la mobilité, stimulent expression et production de MMP9 des ?HL60 et semblent diminuer l effet stimulant des HCSMC sur la production de MMP9 par ?HL60. Les HCSMC expriment et/ou sécrétent certains cytokines (IL8, IL6, IL1?, CCL2, CXCL12) impliquées dans l athérosclérose. En conclusion, l interaction ?HL60/HCSMC entraîne une augmentation de production de MMP9, modulée par les ERO, et une stimulation de mobilité des ?HL60. Les cytokines inflammatoires impliquées dans l athérosclérose et exprimées par les HSCMC sont des acteurs potentiels dans la réponse sécrétoire et/ou migratoire des ?HL60.During atherosclerosis leucocytes invade the vascular wall, inducing inflammation and production of oxidative stress by reactive oxygen species (ROS). We tested the effects of an oxidative stress and/or potential interactions between a neutrophil model (?HL60) and human coronary endothelial (HCAEC) or smooth muscle (HCSMC) cells, on ?HL60 mobility and production and activity of cellular metalloproteinases. Cell passaging of the promyelocytic HL60 cell line, differentiated by DMSO in a neutrophil model (?HL60), induces variations in cell mobility and production of MMP9. ROS from biochemical or cellular sources did not modify MMP2 or MMP9 activity in HCAEC or HCSMC. There is no modification of MMPs activities in ?HL60/HCAEC cocultures, but stimulation of ?HL60 MMP9 activity in ?HL60/HCSMC cocultures. Basal migration capacities and N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine -stimulated invasion abilities of ?HL60 increase in presence of HCSMC. Antioxidant enzymes barely change ?HL60 mobility, increase expression and production of ?HL60 MMP9 and seem to reduce the stimulating effect of HCSMC on ?HL60 MMP9 production. HCSMC also express and/or secrete some cytokines (IL8, IL6, IL1?, CCL2, CXCL12) implicated in atherosclerosis. In conclusion, interactions between ?HL60 and HCSMC induce an increase in MMP9 secretion, which is modulated by ROS, and a stimulation of ?HL60 mobility. Expression by HCSMC of inflammatory cytokines implicated in atherosclerosis allows to identify potential candidates responsible for the secretory and/or migratory response of ?HL60.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Molecular basis for cellular effects of naturally occurring polyamines

The naturally occurring polyamines, putrescine, spermidine and spermine, and the analogue cadaverine, induce a dose-dependent histamine release from rat peritoneal mast cells. Spermine was the most active among these polycationic metabolites, followed by spermidine and putrescine. The histamine release was inhibited by a 2 h pretreatment of the cells with pertussis toxin (100 ng/ml), demonstrating the involvement of a pertussis toxin-sensitive GTP-binding regulatory protein during the exocytotic process. Experiments performed with purified Go/Gi proteins reconstituted into phospholipid vesicles showed a direct stimulation of GTPase activity by the polyamines. This direct stimulation of G proteins and the consequent activation of the coupled effectors may represent a new mechanism of action for natural polyamines controlling receptor-dependent processes

Effect of SK&F 96365 on extracellular Ca2+ -dependent O2- production in neutrophil-like HL-60 cells

Store-operated Ca2+ entry is referred to a capacitative current activated by Ca2+ -stores depletion in various non-excitable cells. Neutrophil-like HL-60 cells responded to N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP) by an early O2- production preceded by a [Ca2+]i rise. Cell stimulation in the absence of extracellular Ca2+ resulted in a major reduction of [Ca2+]i rise and O2- production. A purported inhibitor of store-operated Ca2+ entry, SK&F 96365 (1-(beta-(3-(4-methoxy-phenyl)propoxyl)-4-methoxy-phenetyl)- 1H-imidazole hydrochloride), inhibited extracellular Ca2+ -dependent [Ca2+]i rise by 30% but did not alter O2- production. In conclusion, SK&F 96365 did not modify extracellular Ca2+ -dependent O2- production, despite a significant but limited reduction in fMLP-activated membrane Ca2+ fluxes which can be ascribed to store-operated Ca2+ entry. Furthermore, Ca2+ influx is necessary for a full induction and maintenance of the biological response

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation