Dexamethasone and RU24858 induce survival and growth factor receptor bound protein 2, leukotriene B4 receptor 1 and annexin-1 expression in primary human neutrophils

Glucocorticoids are widely used anti-inflammatory medication in diseases like asthma and chronic obstructive pulmonary disease. Glucocorticoids can either activate (transactivation) or inhibit (transrepression) transcription. RU24858 was introduced as a dissociated glucocorticoid and it has been reported to transrepress but not to transactivate. The aim of this study was to compare the effects of RU24858 and dexamethasone in human neutrophils. RU24858 delayed spontaneous neutrophil apoptosis and further enhanced GM-CSF- induced neutrophil survival to a similar extent as dexamethasone. Like dexamethasone RU24858 also reduced CXCL8 and MIP-1α. Unexpectedly however, RU24858 increased the expression of the glucocorticoid-inducible genes BLT-1, Annexin-1 and Grb-2 in neutrophils to a similar level as seen with dexamethasone. We have shown here that dexamethasone and RU24858 both increase Grb-2, BLT1 and Annexin-1 expression and inhibit CXCL8 and MIP-1α production. This suggests that RU24858 was not able to dissociate between transactivation and transrepression in human neutrophils but enhanced neutrophil survival. © the author(s), publisher and licensee Libertas Academica Ltd

Histone deacetylase inhibitors induce apoptosis in human eosinophils and neutrophils

BACKGROUND: Granulocytes are important in the pathogenesis of several inflammatory diseases. Apoptosis is pivotal in the resolution of inflammation. Apoptosis in malignant cells is induced by histone deacetylase (HDAC) inhibitors, whereas HDAC inhibitors do not usually induce apoptosis in non-malignant cells. The aim of the present study was to explore the effects of HDAC inhibitors on apoptosis in human eosinophils and neutrophils. METHODS: Apoptosis was assessed by relative DNA fragmentation assay, annexin-V binding, and morphologic analysis. HDAC activity in nuclear extracts was measured with a nonisotopic assay. HDAC expression was measured by real-time PCR. RESULTS: A HDAC inhibitor Trichostatin A (TSA) induced apoptosis in the presence of survival-prolonging cytokines interleukin-5 and granulocyte-macrophage colony stimulating factor (GM-CSF) in eosinophils and neutrophils. TSA enhanced constitutive eosinophil and neutrophil apoptosis. Similar effects were seen with a structurally dissimilar HDAC inhibitor apicidin. TSA showed additive effect on the glucocorticoid-induced eosinophil apoptosis, but antagonized glucocorticoid-induced neutrophil survival. Eosinophils and neutrophils expressed all HDACs at the mRNA level except that HDAC5 and HDAC11 mRNA expression was very low in both cell types, HDAC8 mRNA was very low in neutrophils and HDAC9 mRNA low in eosinophils. TSA reduced eosinophil and neutrophil nuclear HDAC activities by ~50-60%, suggesting a non-histone target. However, TSA did not increase the acetylation of a non-histone target NF-κB p65. c-jun-N-terminal kinase and caspases 3 and 6 may be involved in the mechanism of TSA-induced apoptosis, whereas PI3-kinase and caspase 8 are not. CONCLUSIONS: HDAC inhibitors enhance apoptosis in human eosinophils and neutrophils in the absence and presence of survival-prolonging cytokines and glucocorticoids

Gliadin induces neutrophil migration via engagement of the formyl peptide receptor, FPR1

Background Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-Activating and chemoattractant chemokine.We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure. Methods Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-Type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control. Results Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe.We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration. Conclusions Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process. Copyright

LTB4 Is a Signal-Relay Molecule during Neutrophil Chemotaxis

SummaryNeutrophil recruitment to inflammation sites purportedly depends on sequential waves of chemoattractants. Current models propose that leukotriene B4 (LTB4), a secondary chemoattractant secreted by neutrophils in response to primary chemoattractants such as formyl peptides, is important in initiating the inflammation process. In this study we demonstrate that LTB4 plays a central role in neutrophil activation and migration to formyl peptides. We show that LTB4 production dramatically amplifies formyl peptide-mediated neutrophil polarization and chemotaxis by regulating specific signaling pathways acting upstream of actin polymerization and MyoII phosphorylation. Importantly, by analyzing the migration of neutrophils isolated from wild-type mice and mice lacking the formyl peptide receptor 1, we demonstrate that LTB4 acts as a signal to relay information from cell to cell over long distances. Together, our findings imply that LTB4 is a signal-relay molecule that exquisitely regulates neutrophil chemotaxis to formyl peptides, which are produced at the core of inflammation sites

<i>In-vivo</i> intestinal luminal injection of PT-gliadin induces an immediate and considerable neutrophil migration.

<p>(A) Image stills at different time points from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138338#pone.0138338.s002" target="_blank">S1 movie</a>. Intravital microscopy of the duodenum of Lys-GFP mice showed a rapid extravasation and recruitment of neutrophils (bright green) within 1 hour after PT-gliadin luminal administration, which was absent in mice treated with the same volume of PBS. Data are representative of results obtained from eight independent experiments (<i>n</i> = 8 for PT-gliadin, <i>n</i> = 8 for PBS). (B) Quantitative analysis of neutrophil recruitment in response to intestinal luminal PT-gliadin challenge. The green fluorescent channel in the original images was extracted, converted and processed into 16 bit binary images using the Image J software. The number of bright spots, which represents the number of neutrophils in the movies, was calculated with Image J using the Analyze Particles function. Neutrophil recruitment started immediately upon intestinal gliadin exposure, became significant after 15 minutes (<i>P</i> = .006) and increased further over time (<i>P</i> = .019 at <i>t</i> = 30 minutes).</p

Binding of fMet-Leu-Phe and PT-gliadin to FPR1.

<p>Flow cytometry analysis of FITC-labeled gliadin peptide or fMet-Leu-Phe binding to FPR1-transfected or non-transfected HEK293T cells. (A) Kinetic binding of FITC-labeled gliadin synthetic peptide, TLPAMCNVYIPPYCTIVPFG, applied at increasing concentrations (ranging from 1 to 250 μM). Dissociation constant (K_d) was 235.1 μM, and Bmax was 922.4. (B) Kinetic binding of FITC-labeled fMet-Leu-Phe, applied at increasing concentrations (ranging from 1 to 1000 nM). Dissociation constant (K_d) was 27.6 nM, and Bmax was 261.6. (C) Competitive binding assay was performed with FITC-labeled gliadin peptide at 25.6 μM and unlabeled fMet-Leu-Phe at increasing concentrations (ranging from 10 nM to 2 mM). fMet-Leu-Phe caused a displacement of the gliadin peptide from the FPR1-transfected HEK293T cells with an IC50 of 2.04 μM. (D) Competitive binding assay was performed with FITC-labeled fMet-Leu-Phe at 100 nM and unlabeled gliadin peptide at increasing concentrations (ranging from 1 nM to 500 μM). The gliadin peptide was not capable of displacement of the fMet-Leu-Phe from the FPR1-transfected HEK293T cells. Binding to non-transfected cells and FPR1-transfected HEK293T cells is depicted with white and black circles, respectively. Each graph represents data from 3–5 independent experiments.</p

Blocking of FPR1 abrogates neutrophil migration to fMet-Leu-Phe and gliadin.

<p>Pretreatment of neutrophils with cyclosporine H, a specific inhibitor of FPR1, completely abrogated the neutrophil migration induced by PT-gliadin and fMet-Leu-Phe. As expected, LTB₄ induced neutrophil chemotaxis was not affected by cyclosporine H.</p

PT-gliadin attracts neutrophils but does not induce a rapid release of murine KC/CXCL1.

<p>(A) C57BL/6 intestinal segments did not show release of KC during the 2 hours incubation with PT-gliadin (<i>P</i> = NS). The graph represents data obtained from triplicate cultures from three mice. (B-D) EZ-TAXIScan chemotaxis of neutrophils isolated from the bone marrow of C57BL/6 mice toward medium alone, PT-gliadin or fMet-Leu-Phe (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138338#pone.0138338.s003" target="_blank">S2 movie</a>). (B) Images from representative EZ-TAXIScan movies. (C) Image shows the paths of individual cells migrating to PT-water, PT-zein, PT-gliadin (1 mg/mL) or fMet-Leu-Phe (100 nM). The colors progress from red to blue as a function of time. (D) Quantitative analysis of all EZ-TAXIScan assay results of murine neutrophil chemotaxis in response to medium alone, PT-gliadin and fMet-Leu-Phe. The table depicts speed (μm/min) and the chemotactic index (considering speed and distance). Data are representative of results obtained from six independent movies.</p