Estudo do papel dos receptores ativados por protease (PAR)2 E PAR4 na fagocitose e ativação in vitro de macrófagos peritoneais obtidos de camundongos C57BL/6

Abstract

The protease activated receptors (PAR) belong to the family of G protein-coupled receptors and are targets of serine proteases, classified from 1 to 4 depending on the order of discovery. Both PAR2 and PAR4 are expressed in leukocytes, and are involved in the recruitment and activation of these cells. Although the role of the PAR is characterized in inflammation and cell activation models, little is known about the role of these receptors in macrophages during effector functions, for example, phagocytosis and the secretion of inflammatory mediators. Therefore, our objective was to evaluate the role of PAR2 and PAR4 on the phagocytosis of zymosan particles and release of inflammatory mediators in peritoneal macrophages in vitro. For that, macrophages were obtained from C57BL/6 mice, which were previously injected with 6% thioglycollate solution intraperitoneally. After three days, was made the collection of the peritoneal fluid and these cells were pre-incubated with LPS (10 g/ml) 30 minutes before the incubation with PAR2 agonist (SLIGRL-NH2,30 M) or PAR4 agonist (AYPGKF-NH2, 30 M in the absence or presence of the respective selective antagonists (PAR2: ENMD 1068, 30 M; PAR4: TCY-NH2, 30 M); followed by addition of zymosan (10 ug/ml). The evaluation was expressed by Phagocytosis Index (PI) defined as the ratio between the sum of the total particles engulfed in 100 cells counted on the percentage of phagocytic macrophages. The culture supernatant was used for the dosage of nitric oxide (NO), reactive oxygen species (ROS) and cytokine production (TNF- and IL-10). Incubation of macrophages with PAR2 agonist was able to increase the PI, which did not occur when there was a concomitant incubation with antagonist thereof. However, incubation with PAR4 agonist reduced the PI. This reduction was not observed when administered with his antagonist. The co-incubation of PAR2 agonist (30M) with PAR4 agonist (30M) did not alter the PI. The PAR2 activation in vitro or PAR4 was able to increase NO production after 24 hours of incubation. However, after 48 hours of incubation, there was a reduction in the production of this mediator. The PAR2 activation (30M) did not alter the levels of ROS 24 and 48 after incubation. Nevertheless, in the presence of PAR4 agonist (30 M) an increase in ROS production was observed in both times analyzed. Incubation of macrophages with SLIGRL-NH2 (30M) or AYPGKF-NH2 (30M) did not affect TNF- production, but PAR2 agonist increased IL-10 production in 24 hours, whereas the PAR4 agonist reduces the production of cytokine after 4 hours of incubation. In conclusion, the data suggest a regulatory role of PAR2 and PAR4 in the phagocytic phenomenon of macrophages and in the production of mediators pro and anti-inflammatory essential for controlling the effector response of these cells.