Efeito da Migalina sobre atividade inflamatória com diferentes estímulos em linhagens de macrófagos J774 e derivados de medula óssea

Abstract

Natural polyamines putrescine, spermine and spermidine are essential for cell growth and proliferation, protein and nucleic acid synthesis, cell matrix repair, adhesion and signaling processes. Mygalin was isolated from hemocytes of the spider Acanthoscurria gomesiana and identified as a bis-acylpolyamine analogue to spermidine, possessing microbicidal activity. Macrophages are innate immune response cells fundamental in the inflammatory process and are programmed to detect molecular patterns associated with pathogens. Immune cell surface molecules and cytokines are the key components for the achievement of the inflammatory response. Due to the limited knowledge of the role of Mygalin in the immune response, we investigated its immunomodulatory function in macrophages J774 and macrophages derived from C57BL / 6 mouse marrow MODM against several stimuli in different assays. The cells were either preincubated with Mygalin 40-160 μg/ml for 2 hours prior to stimulation or not with LPS 100 ng/ml, Toxoplasma gondii antigen 10-25-50 μg/ml, IFN-γ 1 ng/ml and Zimosan 10 μg/ml After 24 hours, hydrogen peroxide production was examined by the fluorimetric method with and without PMA (200 nM) and the alteration of the expression of the surface molecules by flow cytometry FACS. Supernatants from the J774 and MODM assays were harvested for quantify production of IL-12p40 ELISAand nitrite Griess. Treatment of cells with Mygalin and Toxoplasma gondii didn’t induce IL-12p40 and NO production. In J774 IFN-γ didn’t change the amount of NO in the control, but the LPS increased even in the presence of Mygalin and the same happened with LPS+IFN-γ. Zimosan increased NO production in J774, but IFN- γ reduced it. In MODM independent of the stimulus there was no alteration of the NO synthesis compared to control. Mygalin didn’t increase the production of IL-12p40 when the cells were stimulated by LPS and LPS+IFN-γ. Zimosan induced the synthesis of IL-12p40, but with IFN-γ the production reduced. We evaluated the effect of Mygalin on the production of hydrogen peroxide in MODM that Migalina reduced H2O2 production in the presence of PMA. Regarding the expression of CD11b: in J774 the LPS and LPS+IFN-γ decreased expression; F4 / 80: in MODM the LPS increased its synthesis; CD86: in J774 the LPS decreased expression even with IFN-γ, and in MODMall stimuli reduced synthesis even with Mygalin; CD284: no change in J774cells; CD206: LPS and LPS+IFN-γ there was a slight increase of the synthesis.The mechanisms involved are being analyzed for a better characterization ofthe role of macrolide in the macrophage.