Papel da plasmina na migração de células inflamatórias

Abstract

The Plasminogen/Plasmin (Plg/Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair and inflammation. Although the capacity of Plasmin to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we investigate the capacity of Pla to induce cell migration in vitro and in vivo and the role of mitogen-activated protein kinase (MAPK) ERK1/2, protease activated receptor-1 (PAR-1) and CCL2/CCR2 axis in this process. With this purpose, murine fibroblasts and macrophages were treated with Pla (2µg/mL) at different times, or pretreated with the U0126 (MEK/ERK inhibitor), Leupeptin (serine protease inhibitor) or Tranexamic Acid (lysine analog) 1h before and during treatment with Pla. The cells were processed for analysis of cell migration or analysis of the activation of ERK1/2 by western blot. In vivo experiments were carried out in BALB/C mice which were challenged by intrapleural injection of Pla (2g) and the cells present in the pleural cavity were collected at different times and analyzed for total and differential leukocyte counts, activation of ERK1/2 and NF-κB pathways, production of chemokines and cytokines and immunophenotyping by flow cytometry. Specific inhibitors for MEK/ERK (U0126- 2mg/Kg/i.pl.), serine protease (Leupeptin- 100g/ cavity/i.pl.), protease activated receptor-1 (PAR1- SCH79797 5mg/kg/i.p.) and the chemokine receptor CCR2 (RS504393- 2mg/kg/i.pl.) were administered 1 h before Pla and the cells of the pleural cavity were collected 48h after and processed for total and differential leukocyte counts and western blot for P-ERK and P-IkB Pleural levels of IL-1, IL-6, TNF- and CCL-2 were measured by ELISA. Plasmin induced migration of fibroblasts and murine macrophages in vitro dependent of the MEK/ERK pathway, its proteolytic activity and its lysine binding sites. The injection of plasmin in the pleural cavity of mice induced a time-dependent influx of mononuclear cells associated with increased phosphorylation of ERK1/2 and IκB- and increased levels of CCL2 and IL-6 in the pleural exudates. The inhibition of protease activity of plasmin by using Leupeptin, or the use of PAR- 1 antagonist (SCH79797) was able to abolish the recruitment of mononuclear cells and ERK1/2 and IκB- phosphorylation. In agreement with the in vitro results, the inhibition of MEK/ERK abolished the influx of mononuclear cells and release of MCP-1/CCL2, both induced by plasmin. Experiments performed to confirm the involvement of MCP-1/CCL2 in cell migration induced by plasmin, showed that mice deficient in CCR2 (CCR2-/-) are refractory to the recruitment of mononuclear cells induced by this protease and the use of a CCR2 antagonist (RS504393) abolished plasmin induced migration in vivo and in vitro. We conclude in this study that plasmin is able to induce in vivo migration of mononuclear cells dependently of PAR-1 activation of MEK/ERK/NF-κB pathway resulting in the release of MCP-1/CCL2 and CCR2 activation.