Molecular mechanisms of endogenous glucocorticoids on neutrophil traffic: actions on SDF-1alfa/CXCR-4 and IL-17/IL-23/G-CSF axis

Abstract

Our research group has investigated the mechanisms of actions of endogenous glucocorticoids (eGC) on neutrophil mobilization during basal and inflammatory conditions evoked by different stimulus. We have shown that eGC control the interactions of circulating neutrophils with the microvascular endothelium, involving distinct molecular actions on adhesion processes in each cell type. In addition, we recently demonstrated that eGC modulate the neutrophil mobilization from the bone marrow into peripheral blood in physiological conditions, and that annexin A1, a protein secreted by several cells under glucocorticoids stimulation, mediates this process, modulating the phagocytic activity of bone marrow macrophages and the SDF-1alpha/CXCR-4 axis.The present study keeps on these investigations, by studying the neutrophil traffic under different concentrations of eGC (RU 38486 or ACTH treatments) or under inflammatory reaction (LPS, i.p.), focusing on SDF-1alpha/CXCR-4, IL-17/IL-23/G-CSF, and CD49d, CD18 and CD62L adhesion molecules expressions by neutrophils. It will be employed Male Balb/c mice (2 months old) treated with RU 38486 (glucocorticoid receptor antagonist) or vehicle, and ANXA1 (ANXA1-/-) knockout animals and respective wild type. In addition, these animals will be treated with ACTH (250µg/kg; i.p.) or LPS (50 µg/kg; i.p.) to induce eGC secretion. Bone marrow granulocytes and circulating neutrophils will be quantified by optical microscopy, complemented by flow cytometry assays, using Ly6G labelling; CD49d, CD62L and CD18 adhesion molecule and SDF-1 alpha receptor, CXCR-4, expressions by bone marrow granulocytes and peripheral neutrophils will be measured by flow cytometry; SDF-1alpha, IL-17, IL-23, G-CSF in bone marrow perfusate and plasma, as well as plasma corticosterone, will be quantified by enzyme-linked immunosorbent assay (ELISA) .It is noteworthy to mention that this project will strengthen the collaboration with Dr. Mauro Perretti, William Harvey Institute of London, and Dr. Sara Rankin, Imperial College of London. (AU)