Effects Of Leukotriene D4on Activation Of BV2Microglial Cells

Background:Cysteinyl leukotrienes (CysLTs, including LTC4, LTD4and LTE4), an important class of inflammatory mediators derived from arachidonic acid metabolism via5-lipoxygenase pathway, is involved in regulating many pathophysiological processes in vivo by activating cysteinyl leukotriene receptors1and2(CysLT1and CysLT2).Previous researches found that in the course of cerebral ischemia, the production of cysteinyl leukotrienes in brain increased, which mediated neuronal damage after ischemia; giving injections of leukotriene D4into cortex of mice induced neuronal injury. However, in vitro cellular experiments showed that leukotriene D4failed to induce neuronal injury. The inconsistency of the obtained in vivo and in vitro experimental results made us speculate that the effect of leukotriene D4on inducing neuronal injury in vivo may be mediated by other cell types, such as microglia, which is responsible for the regulation of CNS inflammation, in the brain. In response to cerebral ischemia or encephalitis, microglia is activated promoptly to become the activated microglia, which plays a vital role in the progression of post-ischemia inflammation. However, whether CysLTs is involved in inducing activation of microglia or the mechanism of CysLTs-induced activation of microglia remain to be clarified.Objectives:To observe the effects of leukotriene D4on activation murine BV2microglia, and to investigate the mechanisms underlying leukotriene D4-induced activatin of microglia.Methods:1. Rabbits were immunized with KLH-coupled CysLT1peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by ELISA, and the specificity of the pAb was tested by antigen blockade;2. The expression of CysLT1and CysLT2mRNA and protein were determined by RT-PCR, Western blotting; the subcellular localization CysLT1and CysLT2receptors was detected by immunostaining;3. After treatment with various concentrations (0-100nM) of leukotriene D4(LTD4) for24h, BV2cells were examined under an optical microscopy to observe the morphology change; or using CCK kit to detect cell viability; or using fluorescent bead tracking method to determine microglial phagocytosis.4. Before exposure to LTD4for24h, BV2cells were pretreated with CysLT1receptor selective antagonist Montelukast (1-1000nM), CysLT2receptor selective antagonist HAMI3379(10-10000nM) and CysLT1/CysLT2receptor dual antagonist BAY u9773(1-1000nM) for30minutes. Microglial phagocytosis and mRNA expression of proinflammatory cytokines TNF-a, IL-1(3and IL-6were determined by fluorescent bead tracking method and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).Results:1. We successfully prepared a rabbit anti-mouse CysLT1receptor polyclonal antibody, ELISA tests showed that the pAb had a titer as high as1/32728, and was not cross-reacted with the antigens of CysLT2receptor and GPR17.2. BV2cells have CysLT1and CysLT2receptors both at the mRNA and protein expression levels; immunostaining showed that CysLT1and CysLT2receptors were mainly located on the cell membrane;3. The morphology and cell viability of BV2cells were unaffected by exposure to LTD4for24h;4. Twenty-four-hour exposure to LTD4significantly enhanced the phagocytic activity of BV2cells, and increased IL-6mRNA expression, but did not induce TNF-α, IL-1β mRNA upregulation;5. Pretreatment with CysLT1receptor selective antagonist Montelukast (100nM), CysLT2receptor selective antagonist HAMI3379(100,1000and10000nM) and CysLT,/CysLT2dual antagonist BAY u9773(100nM) for30min significantly increased LTD4(100nM)-induced activation of phagocytosis; BAY u9773(1nM) antagonized the effects of LTD4(100nM) on activation of phagocytosis;6. CysLT1receptor selective antagonist Montelukast (1nM), CysLT1/CysLT2receptor dual antagonist BAY u9773(1nM and10nM) antagonized LTD4(100nM)-induced upregulation of IL-6mRNA. CysLT2receptor selective antagonists HAMI3379(10-10000nM) has no effect on abrogating LTD4-induced increase of IL-6mRNA expression.Conclusions:LTD4is able to induce activation of murine BV2microglia. The profile of LTD4-activated BV2cells was characterized as enhanced phagocytic activity and upregulation of IL-6mRNA; LTD4-induced upregulation of IL-6mRNA was mediated by the CysLT1receptor; the mechanisms of LTD4-induced enhancement of microglial phagocytosis need further investigation.