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Monosodium urate-crystal-stimulated phospholipase D in human neutrophils.

ABSTRACT: Protein kinase Calpha (PKCalpha) and small GTPases of the Rho and ADP-ribosylation factor (Arf) family are implicated in the regulation of phospholipase D1 (PLD1) activity. Although they are involved in fMet-Leu-Phe (fMLP)-mediated PLD activation, their role in monosodium urate (MSU)-stimulated PLD1 activity in human neutrophils is not clear. The translocation of PKCalpha, RhoA and Arf from the cytosol to the membranes was monitored. fMLP induced a cytochalasin B (CB)-dependent recruitment of Arf, RhoA and PKCalpha to neutrophil membranes. CB also increased the activation of PLD 10-fold. In contrast with fMLP, MSU stimulated a sustained and time-dependent relocalization of Arf and PKCalpha, but not of RhoA, to the membrane fraction. MSU-stimulated PLD was activated with a time course preceding membrane recruitment of Arf and PKCalpha in the absence of CB. Furthermore, MSU-induced PLD activation and the membrane recruitment of PKCalpha, but not that of Arf, were inhibited by CB. An anti-FcgammaRIIIB antibody, VIFcRIII, prevented the membrane relocalization of Arf and PKCalpha and the stimulation of the levels of tyrosine phosphorylation and of PLD activity induced by MSU. Erbstatin and ST-638, two inhibitors of tyrosine kinases, inhibited the MSU-induced translocation of Arf and PKCalpha but not MSU-induced tyrosine phosphorylation and PLD activation. Furthermore MSU crystals did not cause the tyrosine phosphorylation of PLD1. The present study indicates that soluble and particulate agonists show selectivity in inducing the translocation of RhoA in neutrophils and that the ability of MSU to increase PLD activation was independent of the membrane relocalization of Arf and PKCalpha.

SUBMITTER: Marcil J

PROVIDER: S-EPMC1219951 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Project description:Background and objectiveBone erosion is common in patients with gout. The role of neutrophil-derived exosomes in gouty bone erosion remains elusive. This study aimed to investigate the functions of the neutrophil-derived exosomes in the development of bone erosion in gout.MethodsNeutrophil-derived exosomes were collected and assessed by transmission electron microscopy and nanoparticle tracking analysis. Cell counting kit-8 assay was applied to evaluate cell viability, and cell apoptosis was assessed by flow cytometry. In addition, quantitative Real-time PCR and Western blotting were used to determine the expression levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL). Neutrophil-derived exosomes were tagged with PKH67. The miRNA expression profiles of exosomes and human fetal osteoblasts (hFOB) were compared using high-throughput sequencing. Functional miRNAs transfected into hFOB after co-incubation with exosomes were selected and validated by preliminary qPCR.ResultsNeutrophil-derived exosomes were stimulated by monosodium urate (MSU). The exosomes could inhibit the viability of the hFOB, and the expression levels of ALP and OPG were down-regulated, while the expression level of RANKL was up-regulated. However, there was no significant difference in the viability of osteoclasts and the expression of nuclear factor of activated T cells 1. Exosomes were observed in the cytoplasm under a confocal microscopy, confirming that exosomes could be taken up by hFOB. In total, 2590 miRNAs were found, of which 47 miRNAs were differentially expressed. Among the delivered miRNAs, miR-1246 exhibited the highest level of differential expression. The viability of hFOB was reduced by miR-1246 mimics and increased by miR-1246 inhibitors. There was no significant difference in hFOB apoptosis rate between the miR-1246 mimic and miR-1246 inhibitor group. MiR-1246 overexpression decreased the expression levels of ALP and OPG, whereas increasing the expression level of RANKL. In contrast, miR-1246 inhibitor increased the expression levels of ALP and OPG, while decreasing the expression level of RANKL. Neutrophil-derived exosomes stimulated by MSU could increase the expression of miR-1246.ConclusionNeutrophil-derived exosomes stimulated by MSU could inhibit the viability of osteoblasts.

Project description:Background:Marantodes pumilum is traditionally used for dysentery, gonorrhea, and sickness in the bones. Previous studies revealed its antibacterial and xanthine oxidase inhibitory activities. Objective:To evaluate the inhibitory effects of three M. pumilum varieties on the secretion of lipopolysaccharide (LPS)- and monosodium urate crystal (MSU)-induced cytokines and plasma prostaglandin E2 (PGE2) in vitro. Materials and Methods:The leaves and roots of M. pumilum var. alata (MPA), M. pumilum var. pumila (MPP), and M. pumilum var. lanceolata (MPL) were successively extracted with dichloromethane (DCM), methanol, and water. Human peripheral blood mononuclear cells and ELISA technique were used for the cytokine assay, whereas human plasma and radioimmunoassay technique were used in the PGE2 assay. Flavonoids content was determined using a reversed-phase high-performance liquid chromatography. Results:DCM extract of MPL roots showed the highest inhibition of LPS-stimulated cytokine secretion with IC50 values of 29.87, 7.62, 5.84, 25.33, and 5.40 ?g/mL for interleukin (IL)-1?, IL-1?, IL-6, IL-8, and tumor necrosis factor (TNF)-?, respectively; while that of plasma PGE2 secretion was given by DCM extract of MPP roots (IC50 31.10 ?g/mL). Similarly, the DCM extract of MPL roots demonstrated the highest inhibition against MSU-stimulated IL-1?, IL-1?, IL-6, IL-8, TNF-?, and PGE2 secretion with IC50 values of 11.2, 8.92, 12.29, 49.51, 9.60, and 31.58 ?g/mL, respectively. Apigenin in DCM extracts of MPL (0.051 mg/g) and MPP (0.064 mg/g) roots could be responsible for the strong inhibitory activity against IL-1?, IL-6, TNF-?, and PGE2. Conclusion:The results suggested that DCM extracts of MPL and MPP roots are potential anti-inflammatory agents by inhibiting the secretion of LPS- and MSU-stimulated pro-inflammatory cytokines and PGE2. SUMMARY:Amongst 18 tested extracts, DCM extracts of MPL and MPP roots remarkably inhibited LPS- and MSU-stimulated pro-inflammatory cytokines and PGE2 secretionPhytochemical analysis was performed for the active extracts using RP-HPLC systemThe presence of flavonoids particularly apigenin could be responsible for the anti-inflammatory activity. Abbreviations used: BSA: Bovine serum albumin, COX-2: Cyclooxygenase-2, CPM: Count per minute, DAMP: Danger-associated molecular pattern, DCM: Dichloromethane, DMSO: Dimethyl sulfoxide, ELISA: Enzyme-linked immunosorbent assay, FBS: Fetal bovine serum, H2O: Water, HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, HMC-1: Human mast cell-1, HMGB1: High-mobility group box 1, ICAM: Intercellular adhesion molecule, IFN: Interferon, IgG: Immunoglobulin G, IKK: IkB kinase, IL: Interleukin, iNOS: Inducible nitric oxide synthase, LPS: Lipopolysaccharide, MeOH: Methanol, MPA: Marantodes pumilum var. alata, MPL: Marantodes pumilum var. lanceolata, MPP: Marantodes pumilum var. pumila, MSU: Monosodium urate, MTT: Methylthiazole tetrazolium, NF-?B: Nuclear factor-kappa B, NLR: NOD-like receptor, NLRP3: NLR family pyrin domain containing protein 3, NO: Nitric oxide, NOD: Nucleotide-binding oligomerization domain, NSAID: Nonsteroidal anti-inflammatory drug, PAMP: Pathogen-associated molecular pattern, PBMC: Peripheral blood mononuclear cell, PBS: Phosphate buffered saline, PGE2: Prostaglandin E2, PMACI: Phorbol-12-myristate 13-acetate and calcium ionosphere A23187, PRR: Pathogen recognition receptor, PTFE: Polytetrafluoroethylene, RIA: Radioimmunoassay, RIG: Retinoic acid-inducible gene I, RLR: RIG I-like receptor, RP-HPLC: Reversed-phase high-performance liquid chromatography, RPMI-1640: Roswell Park Memorial Institute-1640, TLR: Toll-like receptor, TNF: Tumor necrosis factor, VCAM: Vascular cell adhesion molecule.

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Monosodium urate-crystal-stimulated phospholipase D in human neutrophils.

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