Connections of annexin A1 and translocator protein-18 kDa on toll like receptor stimulated BV-2 cells.

BACKGROUND: Annexin A1 (ANXA1) and Translocator Protein-18KDa (TSPO) down-regulate neuroinflammation. We investigated the role of recombinant ANXA1 (rANXA) on TSPO functions on Toll Like Receptor (TLR) activated microglia. METHODS: BV-2 cells (murine microglia), were stimulated by E. coli Lipopolysaccharide (LPS) and treated with rANXA1 in order to measure TSPO expression and inflammatory parameters. Anti-sense ANXA1 and TLR4 and TSPO shRNA, as well as pharmacological treatments, were employed to assess the mechanisms involved. RESULTS: LPS-stimulated BV-2 cells caused overexpression of TSPO, which was inhibited by: pharmacological blockade of TLR4 or TLR4 mRNA silencing; inhibition of myeloid differentiation primary response gene 88 (MyD88) dimerization; or blocking of nuclear factor κB (NF-κB) activation. rANXA1 treatment impaired LPS-induced TSPO upregulation by down-modulating MyD88 and NF-κB signaling; the effect was abolished by WRW4, an antagonist of formyl peptide receptor 2 (FPR2). rANXA1 treatment also downregulated interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNFα) secretion in LPS-stimulated BV-2 cells. TSPO knockdown in BV-2 cells augmented LPS-induced TNFα secretion and abolished the inhibitory effect of rANXA1 on TNFα secretion evoked by LPS. CONCLUSIONS: exogenous ANXA1 down-modulates LPS-induced TSPO via MyD-88/NF-κB pathways, and constitutive TSPO is pivotal for the control of ANXA1 on TNFα secretion. TSPO actions may be involved with the mechanisms of ANXA1 on inflammatory brain diseases.

Involvement of the Rho-kinase/myosin light chain kinase pathway on human monocyte chemotaxis induced by ATL-1, an aspirin-triggered lipoxin A4 synthetic analog.

Lipoxins (LX) are arachidonic acid metabolites able to induce monocyte chemotaxis in vitro and in vivo. Nonetheless, the signaling pathways mediating this process are yet unclear. In this study, we have investigated the mechanisms associated with human monocyte activation in response to 15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATL-1), a stable 15-epi-LXA4 analog. Our results demonstrate that ATL-1-induced monocyte chemotaxis (10-300 nM) is inhibited by pertussis toxin, suggesting an effect via the G-protein-linked LXA4 receptor. Monocytes stimulated with the analog presented an increased ERK-2 phosphorylation, which was reduced by PD98059, a selective inhibitor of the MEK 1/2 pathway. After exposure of the cells to ATL-1, myosin L chain kinase (MLCK) phosphorylation was evident and this effect was inhibited by PD98059 or Y-27632, a specific inhibitor of Rho kinase. In addition, Y-27632 abolished ERK-2 activation, suggesting that the MAPK pathway is downstream of Rho/Rho kinase in MLCK activation induced by ATL-1. The specific MLCK inhibitor ML-7, as well as Y-27632, abrogated monocyte chemotaxis stimulated by the analog, confirming the central role of the Rho kinase/MLCK pathway on ATL-1 action. Together, these results indicate that ATL-1 acts as a potent monocyte chemoattractant via Rho kinase and MLCK. The present study clarifies some of the mechanisms involved on the activation of monocytes by LXs and opens new avenues for investigation of these checkpoint controllers of inflammation.