Effects Of Eysteinyl Ieukotriene Receptors On Phagocytosis In BV2Microglial Cells

Objectives:The aim of this study was to determine the effects of cysteinyl leukotriene receptors (CysLT1R and CysLT2R) on phagocytosis in mouse BV2microglial cells, and the possible interactions between two subtypes of CysLT receptors.Methods:1. BV2cells were stimulated with CysLT receptor agonists (LTD4and NMLTC4) and microglial activators (LPS and IFN-y), and the phagocytosis of BV2cells was evaluated by immunofluorescence staining and flow cytometry.2. After pretreatment for30min with the selective CysLT1R antagonist montelukast and the selective CysLT2R antagonist HAMI3379, their effects on phagocytosis induced by the agonists and activators were evaluated.3. Western blotting was used to detect expression changes in CysLT1R and CysLT2R induced by the activators LPS and IFN-y, and to evaluate the influence of the antagonists montelukast and HAMI3379.4. Immunofluorescence staining methodwas used to detect CysLTs receptor distribution changes in BV2cells. At the defined time-point, the changes were preliminarily confirmed by confocal laser scanning microscopy.Results:1. Immunofluorescence staining and flow cytometry showed that the CysLT receptor agonists LTD4and NMLTC4as well as and the microglial activators LPS and IFN-y significantly enhanced the phagocytosis of BV2cells.2. Montelukast and HAMI3379inhibited the enhanced phagocytosis of BV2cells induced by the agonists and activators. Montelukast (0.1μmol/L) and HAMI3379(1μmol/L) significantly decreased LTD4(100nmol/L)-induced activation of phagocytosis. NMLTC4-induced activation of phagocytosis was inhibited by montelukast and HAMI3379, and montelukast was more effective than HAMI3379. Both montelukast (0.1-1μmol/L) and HAMI3379(1μmol/L) also inhibited LPS-induced activation of phagocytosis.3. The activators LPS and IFN-y could up-regulate the CysLT1R and CysLT2R expression. IFN-y only showed an increasing trend of CysLT1R expression, but significantly increased the CysLT1R expression. On the other hand, LPS significantly increased the expression of both CysLT1R and CysLT2R, and this effect was reversed by montelukast and HAMI3379.4. Immunofluorescence staining showed that activation of BV2cells resulted in changes in intracellular distribution of CysLT1R and CysLT2R. The co-localization of CysLT1R and CysLT2R was determined by immunofluorescence staining and preliminarily confirmed by confocal laser scanning microscopy.Conclusions:1. Both CysLT1R and CysLT2R regulate the activation of BV2cells. The activation of BV2cells has been characterized by increased phagocytic activity and up-regulation of CysLT1R and CysLT2R expression, which can be inhibited by CysLTreceptor antagonists.2. The interactions between CysLT1R and CysLT2R may exist, but need further investigation.