Regulatory Role Of The MGluR Ⅰ Activation On The Adhesion Of Neutrophils To Endothelial Cells

Glutamate, a major excitatory neurotransmitter in the brain, presents abundantly in the mammalian central nervous system (CNS),and plays a variety of important physiological roles by acting on the glutamate receptors. Excessive activation of glutamate receptors, may however be responsible for a wild variety of neurotoxic effects in the CNS.Recently, sufficient evidence has been provided for the presence of glutamate receptors in peripheral tissues, while the function of these receptors is largely unknown. Earlier experiments in this laboratory indicated that excessive activation of the ion channel glutamate receptor, N-methyl-D-aspartate receptor (NMDAR), in neutrophils promoted the adherence of neutrophils to endothelial cells, and aggravated the acute lung injury. In this study, we search for the evidence of the expression of group I metabotropic glutamate receptors (mGluR I,include mGluRl and mGluR5)in human neutrophils and endothelial cells. The effect of mGluR I activation on the adhesion between neutrophils and endothelial cells is investigated,which may provide novel pathological mechanism and therapeutic approaches for the prevention of acute lung injury (ALI).Methods:Ficoll-Hypaque density gradient centrifugation was used to separate neutrophils through neutrophils separating medium and leukomonocytes separating medium. Immunocytochemical method and realtime-PCR were used to detect the expression of mGluR I (mGluRl and mGluR5)in neutrophils and human normal umbilical vein endothelial cells line(HUVE-12).Different concentrations(10-8～10-4 mol/L) of mGluR I specific agonist, S-3,5-dihydroxy-phenylglycine (S-DHPG), were applied to the incubated neutrophils or endothelial cell at different time points, then the adherence of neutrophils to endothelial cell was observed by colorimetric method. Furthermore, the adherence of neutrophils to endothelial cell after application of the mGluR I specific antagonist,(RS)-alpha-methyl-4-carboxyphenylglycine ((±)-MCPG, 0.5 mmol/L) was also tested. The expression of adhesion molecule CD11a in the neutrophils and ICAM-1 in endothelial cells treated with S-DHPG (10-6mol/L) for 1h was determined by flow cytometry.Results:1.The results confirmed the expression of mGluRl and mGluR5 in human neutrophils and endothelial cells using immunocytochemical methods and realtime-PCR.2. The application of S-DHPG(10-8-10-6mol/L)for 1 h on neutrophils significantly increased the rate of neutrophils adhesion to endothelial cell in a dose-dependant manner, with a maximum effect at 10-6 mol/L (P<0.01).Following time extension (0.5-5 h), the treatment with S-DHPG(10-6 mol/L) on neutrophils increased the rate of neutrophils adhesion to endothelial cell with a maximum effect at 0.5h (P<0.01),without a clear time-dependant manner. The treatment with S-DHPG(10-6 mol/L)for 1 h increased the expression of CD11a in neutrophils (P<0.01)as well.Application of the selected mGluR I antagonist, ((±)-MCPG,0.5 mmol/L) abolished the effect of S-DHPG (10-6 mol/L) on neutrophils adhesion to endothelial cells (P<0.01).3.The treatment with S-DHPG(10-8-10-6 mol/L)for 1 h on endothelial cells dramatically increased the rate of neutrophils adhesion to endothelial cells in a dose-dependant manner, with a maximum effect at 10-6 mol/L (P<0.01).Following time extension (0.5-5 h), the treatment with S-DHPG(10-6 mol/L)on endothelial cell increased the rate of neutrophils adhesion to endothelial cell with a maximum effect at 0.5h (P<0.01),without a clear time-dependant manner. The treatment with S-DHPG(10-6 mol/L)for 1 h increased the expression of ICAM-1 in endothelial cell (P<0.01). Application of the selected mGluR I antagonist, ((±)-MCPG,0.5 mmol/L) abolished the effect of S-DHPG (10-6mol/L) in neutrophils adhesion to endothelial cells (P<0.01).Conclusion:1.Both human neutrophils and endothelial cell line can express mGluRl and mGluR5 subtype2.The activation of mGluR I in neutrophils up-regulates the expression of adhesion molecule CD 11 a, and promotes the adherence of neutrophils to endothelial cells.3.The activation of mGluR I in endothelial cells up-regulates the expression of adhesion molecule ICAM-1,and promotes the adherence of neutrophils to endothelial cells.