The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils

BACKGROUND: Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-κB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils. METHODS: Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis. RESULTS: RU24858 (1 μM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 μM) and dexamethasone (1 μM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival. CONCLUSION: Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone

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

The dissociated steroid RU24858 induces transactivation but not repression in human eosinophils

Syventävä työ ei kirjastoss

The dissociated steroid RU24858 induces transactivation but not repression in human eosinophils

Syventävä työ ei kirjastoss

The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils

Abstract Background Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-κB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils. Methods Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis. Results RU24858 (1 μM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 μM) and dexamethasone (1 μM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival. Conclusion Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.</p

Blocking of FPR1 inhibits neutrophil migration to gliadin peptides.

<p>Four alpha-gliadin synthetic peptides that displayed a chemotactic response were elected and tested in the presence of cyclosporine H, a specific inhibitor of FPR1. Blocking of FPR1 inhibited neutrophil chemotaxis to these peptides.</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