Modulação da mieloperoxidase e elastase de neutrófilos pela aminoguanidina

Abstract

Neutrophils represent the organism´s first line of defense against invading microorganisms, infected cells with viruses and tumor cells. In healthy humans, they constitute the most abundant population of white blood cells. The recognition of microbial components by neutrophils initiate phagocytosis, generation of reactive oxygen species (ROS) and release of granular proteins, such as myeloperoxidase (MPO) and elastase (NE). These ROS and enzymes act as microbicidal agents and contribute to the inflammatory processes. In a previous study, we found an increase in phagocyte NADPH oxidase system activity (NOX2) and generation of ROS by neutrophils of diabetic rats and non-diabetics treated with aminoguanidine (AG), a known inhibitor of the formation of advanced glycation end products (AGEs), which resulted in an increased microbicidal activity of these cells. Our aim is to elucidate the mechanism of action by which AG is acting in our model. This way, this study evaluated the hypothesis that AG would be stimulating degranulation of neutrophils, therefore, we studied the modulation of MPO and NE, the main enzymes present in azurophilic granules of neutrophils. For this, we used human neutrophils isolated from peripheral blood collected by venipuncture from healthy volunteers. Neutrophils were incubated or not with AG 0,5 mM for a 18 hour period in cell culture incubator. MPO activity was evaluated through the formation of HOCl and luminol-amplified chemiluminescence. NE activity was evaluated through the formation of the product of p-nitroanilide reaction, by spectrophotometry. We also evaluated the expression of MPO and NE by western blotting and immunostaining. As control of reactions was used inibitor of MPO (azide) and of NE (trypsin inhibitor from Glycine max (soybean) - (STI)). The neutrophil isolation allowed the achievement of a cell population with over 95% of neutrophils, with viability greater than 99%. Our results showed that AG was not capable of influencing the chlorination and basal activity of MPO, however, an increase of ROS by the treatment with AG after stimulation was noted. We also noted that NE activity remained unaltered by the treatment with AG. In addition, AG did not affect expression and distribution of MPO and NE. These results together lead us to suggest that AG does not influence directly the process of neutrophils degranulation, as this process is a mechanism used by phagocytes in order to create a fully hostile environment for invading pathogens. Apparently, the increase of microbicidal activity of neutrophils treated with AG is not via NE or MPO in the classical activity of this enzyme, in other words, in the production of HOCl. However, we observed a significant response in MPO activity, allowing us to propose various activities for this enzyme, either as a oxidative activity that can contribute to the killing or as an antioxidant activity, and therefore may protect other antimicrobial granular proteins from oxidative damage.