3 resultados para CXCL8

em Aston University Research Archive


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The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 µM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

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Background: Human rhinoviral infections are major contributors to the healthcare burden associated with acute exacerbations of asthma. We, and others have recently demonstrated that rhinovirus (RV)-induced inflammatory responses are mediated by multiple signalling mechanisms, such as IL-1/MyD88 (1) and TLR3/RIGI (2). We have also previously published work showing that TLR signalling is effectively inhibited by phosphatidylserine-containing liposomes (SAPS), through the disruption of membrane microdomains (3). Evidence has also suggested that membrane microdomains may influence infections with RV. In this study, we explored the ability of SAPS to modulate responses to the natural viral pathogens, RV-1B and RV-16. Method: The immortalized bronchial epithelial cell line, BEAS-2B or primary bronchial epithelial cells were infected with RV-1B or RV-16 at a TCID50/ml of 19107 for 1 h. Immediately following infection, various concentrations of SAPS were added and changes in cytokine release were measured at 24 h. SAPS remained present throughout. Type I and III interferon (IFN) expression and rates of viral replication were measured by quantitative PCR. Virus quantification was also performed using a viral CPE assay, and IFN signalling was measured by western blot. Liposome stability was characterised and intracellular trafficking of fluorescently labelled SAPS in BEAS-2B cells was investigated using confocal microscopy. For in vivo studies, female wt Balb/c mice were pre-treated with SAPS for 2 h prior to infection with RV as previously described and changes in BAL cell number, BAL cytokine production and viral replication were quantified (4). Results: Characterisation of SAPS liposomes by mass spectrometry showed no obvious signs of oxidation over the time period tested, and liposome size remained constant. Preliminary confocal studies revealed that SAPS was rapidly internalised within the cell and was found to associate with intracellular compartments such as the early endosome and golgi. Viral infected BEAS-2B cells co-incubated with SAPS, showed notably impaired responses to RV as assessed by release of CXCL8 and CCL5. SAPS also reduced RV-induced IFNb production and STAT-1 phosphorylation, without significantly influencing viral replication rates. Modest increases in viral particle production were only observed at 48 and 72 h time points. Suppression of viral-induced cytokine production was also observed in primary bronchial epithelial cells and pilot in vivo studies showed that SAPS results in reduced KC production at 24 h post viral infection, and this was associated with reduced neutrophil numbers within the BAL fluid. Conclusion: Our data demonstrates a potential means of modulating inflammatory responses induced by human rhinovirus.

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Papillon-Lefévre syndrome is a rare, inherited, autosomal-recessive disease, characterized by palmoplantar keratosis and severe prepubertal periodontitis, leading to premature loss of all teeth. Papillon-Lefévre syndrome is caused by a mutation in the cathepsin C gene, resulting in complete loss of activity and subsequent failure to activate immune response proteins. Periodontitis in Papillon-Lefévre syndrome is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, although mechanistic pathways remain to be elucidated. The aim of this study was to characterize comprehensively neutrophil function in Papillon-Lefévre syndrome. Peripheral blood neutrophils were isolated from 5 patients with Papillon-Lefévre syndrome, alongside matched healthy control subjects. For directional chemotactic accuracy, neutrophils were exposed to the chemoattractants MIP-1α and fMLP and tracked by real-time videomicroscopy. Reactive oxygen species generation was measured by chemiluminescence. Neutrophil extracellular trap formation was assayed fluorometrically, and proinflammatory cytokine release was measured following overnight culture of neutrophils with relevant stimuli. Neutrophil serine protease deficiencies resulted in a reduced ability of neutrophils to chemotax efficiently and an inability to generate neutrophil extracellular traps. Neutrophil extracellular trap-bound proteins were also absent in Papillon-Lefévre syndrome, and Papillon-Lefévre syndrome neutrophils released higher levels of proinflammatory cytokines in unstimulated and stimulated conditions, and plasma cytokines were elevated. Notably, neutrophil chemoattractants MIP-1α and CXCL8 were elevated in Papillon-Lefévre syndrome neutrophils, as was reactive oxygen species formation. We propose that relentless recruitment and accumulation of hyperactive/reactive neutrophils (cytokines, reactive oxygen species) with increased tissue transit times into periodontal tissues, alongside a reduced antimicrobial capacity, create a locally destructive chronic inflammatory cycle in Papillon-Lefévre syndrome.