The Effect Of LTB₄-BLT2Pathway On Mouse Dendritic Cells

BackgroundLeukotrienes, the important inflammatory mediators in the body, have already beenconfirmed to participate in occurrence and development of many immune-relateddiseases. Many researches of leukotrienes were carried out in the fields of chronicinflammation of respiratory system, for example asthma. In inflammatory mediatorsrelated immune disease in other tissues or organs, such as Allergic Rhinitis,Ulcerative Colitis, Glomerulonephritis, Rheumatoid Arthritis, lipid mediatorsleukotrienes were significantly higher than normal groups. Therefore, drugstargeting leukotrienes may provide a new direction for the diagnosis and treatmentof these diseases.The leukotrienes are derived from membrane phospholipids. Leukotriene B₄（LTB₄）,5（S）,12（R）-dihydroxy-6（E）,8（E）,10（E）,14（E）-tetraene eicosanoic acid, is apotent proinflammatory cytokines and chemokines, exerting strong chemotactic andproinflammatory effects on a variety of cells, such as neutrophils, eosinophils andmonocytes. A number of studies have already confirmed that LTB₄were involved ininflammation-related diseases. Up to now, two subtypes of LTB₄receptors,belonging to the G-protein-coupled receptors （G protein coupled receptor）, has beenfound and called BLT1and BLT2. The different distribution and expressioncharacteristics of the two subtypes suggest that they may mediate different functions.Discovered earlier, BLT1was found associated with many immune-related diseases,such as Kawasaki disease, chronic obstructive pulmonary disease （COPD） and asthma, and there were many researches on mechanism of LTB₄-BLT1pathway;while the researches of BLT2were rather limited.Dendritic cells （DCs）, the most principal antigen presenting cells （APC） in the body,have been a research focus. The main functions of DCs are,â‘ intaking, processingand presenting antigens;â‘¡selective chemotaxis;â‘¢activate na ve T cells. Manystudies have confirmed that significant dendritic cell infiltration could be found ininflammation-related diseases, such as bladder cancer, stomach cancer, colon cancer,arthritis, rhinitis. BLT2were express in DCs, and expression levels wereup-regulated with cell maturation, which may suggest that LTB₄-BLT2receptorpathway may be involved in immune mechanisms of DCs, and may serve aspotential targets for the diagnosis and treatment of inflammation-related diseases.Methods1. Mouse bone marrow-derived mononuclear cells were primarily isolated andcultured, and the differentiation were induced with mouse recombinant granulocytecolony-stimulating factor （Granulocyte Macrophage Colony Stimulating Factor,GM-CSF） and leukocyte interleukin-4（Interleukin4, IL-4）; mature DCs wereobtained by bacterial lipopolysaccharide （Lipopolysaccharides, LPS） stimulation.The cell state and numbers of DCs were determined by morphological observation,and the expressions of surface factors, such as CD11c, CD86and MHC-â…¡, weredetected by flow cytometry.2. The expression of BLT2on mouse DCs were detected by RT-PCR.3. The LTB₄-BLT2pathway was interfered with RNAi. After the pathway wasinterfered and the cells were stimulated with different concentrations of exogenous LTB₄, the chemotaxis of DCs were assessed by innovatively modified MTTcombined with transwell inserts.4. TNF-α and IL-1β expression of mouse DCs and effects of BLT2pathwayblocking were detected by RT-PCR.5. The TNF-α and IL-1β concentrations of cell culture supernatant were determinedby enzyme-linked immunosorbent assay in DCs of LTB₄-BLT2pathway blockinggroup and control group, after stimulation of exogenous LTB₄of differentconcentrations.Results1. About1×10⁶of DCs could be obtained from PBMC isolated from mouse bonemarrow cells after7days of induced culture. With prolonged incubation time, thecells demonstrated prominent protrusions and agglomerate phenomenon of a smallamount of cells. Flow cytometry showed that high level expression of cytokines,such as CD11c, CD86, MHC-â…¡ in mature DCs, and low level expression ofcytokines in immature cells.2. High expression of BLT2were found in mouse DCs. After optimization of siRNAtransfection, the gene expression and protein expression of BLT2could besignificantly inhibited and the LTB₄-BLT2pathway could be blocked.3. LTB₄could significantly enhance the chemotactic ability of normal DCs, whilesiRNA interfering could block BLT2gene expression in DCs and suppresschemotactic ability. The transwell experiments combined with MTT suggested that3concentrations of LTB₄could significantly enhance chemotaxis of DCs in thenon-transfection group. Compared with the control group, low, medium and high concentration groups increased by27.8%,48.4%,71.5%. After blocking BLT2pathway by siRNA, the effect of LTB₄on chemotaxis was significantly inhibited.Compared with the negative control group, inhibition rates of low, medium, highconcentration groups and blank group were59.5%,58.8%,66%and41.2%,respectively.4. Meanwhile, DCs also expressed TNF-α and IL-1β. When the BLT2pathway wasblocked, expressions of the two cytokines at the gene level were also significantlydown-regulated.5. It was confirmed by ELISA that the secretion of TNF-α and IL-1β in normal DCscould be stimulated by LTB₄, and secretion was proportional to the concentration ofLTB₄. After BLT2expression was inhibited by siRNA, the secretion of twocytokines stimulated by LTB₄in the transfected group were significantly suppressed,compared with the control group.Conclusion:Expression of BLT2was found in mouse DCs. The chemotactic ability of DCsstimulated by LTB₄was influenced by BLT2pathway; secretion of the twocytokines in DCs was associated with LTB₄-BLT2pathway. Suppression of geneexpression of BLT2may lead to down-regulation of chemotaxis of DCs andsecretion of inflammatory cytokines. Understanding of its mechanism may providenew targets for the study of inflammation-related drugs.