Participação do ativador do plasminogênio do tipo uroquinase na migração de células inflamatórias

Abstract

Plasminogen (plg) is a zymogen that is cleaved to plasmin (pla) by the tissue-type (tPA) or urokinase-type (uPA) activator. The classical function of the plg/pla system is the dissolution of fibrin clots, but it has also been shown to be important in other biological activities, such as cell recruitment and inflammation. This study investigated the uPA ability to induce cell migration in vitro, using fibroblasts (MEFs) and macrophages (RAW 264.7) cell lines, and in vivo by injecting uPA in the pleural cavity of mice. It also evaluated the role of the mitogen-activated protein kinase ERK1/2, the focal adhesion proteins FAK and Paxillin (Pax), the chemokine CCL2 and its receptor CCR2 in the process, as well as the profile of recruited cells. MEFs and RAW monolayers were scratched and then treated with uPA (1ìg/ml) for different times or pre-treated with U0126 (a MEK1/2 inhibitor) or RS504393 (a CCR2 antagonist). Cells were processed to count the migration to the scratch region, to analyze the phosphorylation of ERK1/2, FAK and Pax by Western Blot and to measure CCL2 levels by ELISA. BALB/C mice received an intrapleural injection of uPA (1ìg) and the cells present in the cavity were harvested at different time points and processed for total and differential count. The uPA treatment induced MEF and RAW migration dependent on MEK/ERK, CCL2/CCR2 and associated with the phosphorylation of focal adhesion kinases FAK and Pax. The intrapleural injection of uPA induced a time-dependent influx of mononuclear cells into the mice pleural cavity associated with increased phosphorylation of ERK1/2, IB-á and FAK and raised CCL2 levels. Importantly, the inhibition of CCR2 abrogated uPA-induced leukocyte influx. Further investigation of the recruited leukocytes by using flow cytometry showed a predominance of macrophages from M2 profile. In conclusion, uPA induces migration of macrophages in vitro and in vivo e those are from M2 profile.