Control of cytosolic glucocorticoid receptor and Annexin-A1 on neutrophil traffic from bone marrow into blood: Adhesion molecule expression and SDF-1α/CXCR-4/CXCR-2

ID Machado1, JR Santin1, V Ferraz-de-Paula1, M Perretti2, SHP Farsky1. 1University of São Paulo, Clinical and Toxicological Analysis, Brazil, 2William Harvey Research Institute, Centre for Biochemical Pharmacology, UK

Introduction: The traffic of neutrophils is a complex process, dependent on coordinated interaction of chemical substances and their receptors, besides perfect cell interactions. We have shown that endogenous glucocorticoids (EG) and Annexin-A1 (ANXA1), a protein induced by glucocorticoid actions, modulate the neutrophil traffic from the bone marrow, but the mechanisms involved are not fully elucidated. Therefore, this study investigated the role of the glucocorticoid cytosolic receptor (GCR) and ANXA1 participation on neutrophil mobilization from bone marrow (BM) into blood (PB), focusing SDF-1α/CXCR-4/CXCR-2 axis and adhesion molecules (CD18, CD49d and CD62L) expressions.

Methods: ANXA1-null (ANXA1-/-) and Balb/C wild-type male were employed. Balb/C mice were treated with vehicle or with the antagonist of GCR (RU38486; RU; 10 mg/Kg, i.p., 28 e 6 h). PB and BM perfusate were collected to quantify the numbers of leukocytes, expressions of CXCR-2, CXCR-4, CD62L, CD49d and CD18 (flow cytometry) and SDF-1α levels (ELISA). Phagocytosis of senescent neutrophils by BM macrophages obtained from all groups of animals was in vitro assessed by optical microscopy.

Results: RU treatment and ANXA1 deficiency accelerated the granulocyte maturation in BM and caused neutrophilia in PB, but only ANXA1-/- animals showed increased number of mature granulocytes in BM (p<0.05). Both GCR blockade decreased the number of granulocytes CD62L+ (p<0.01) in BM, but GCR blockade (p<0.01) and ANXA1 deficiency (p<0.05) increased the number of neutrophils CD62L+ in PB and the expression of molecule CD62L per neutrophils. No alterations on CD49d and CD18 were detected in all groups of animals. GCR blockade increased and reduced the number of granulocytes CXCR-4+ (p<0.001) and CXCR-2+ (p<0.05) in BM, respectively, and deficiency of ANXA1 enhanced both number of cells CXCR4+ (p<0.05) and number of CXCR-2+ (p<0.05) and expression of this latter receptor per cell. In the PB, GCR blockade enhanced the number of CXCR4+ neutrophils (p<0.01) and reduced the number of neutrophils CXCR-2+ (p<0.05) and its expression per cell. Differently, ANXA1-/- animals only presented reduced number of neutrophils CXCR2+ (p<0.01) and expression per cell in the circulation. SDF-1-α levels in the bone marrow was only reduced in ANXA1-/- animals (p<0.05). BM macrophages collected from RU treated (20.3 ± 1.4 %) or ANXA1-/- (32.5 ± 2.5 %) phagocyted less number of senescent neutrophils than macrophages collected from animals treated with vehicle (48.8 ± 3.2 %).

Conclusion: Data here obtained show that EG modulation on neutrophil traffic from BM into PB is dependent on its interaction to GCR and ANXA1 secretion. Nevertheless, the mechanisms of control on SDF-1α/CXCR-4/CXCR-2 axis is different, suggesting that other pathways besides ANXA1 is involved in the EG modulation of neutrophil traffic.

Sources of research support: FAPESP (2010/08402-2; 2010/16828-0).