Regulation of neutrophil functions by platelets

(1)我们已经开发了一个新的设备，可以对platelet aggregates进行sensitively detect the presence of platelet aggregatesplatelet-neutrophil aggregates in whole blood. A pulse laser and A CCD camera are attached to Aflow cytometer, which is an automated reticulocyte analyzer, R-3000 （Sysmex）. Forward scatter intensity and fluorescence intensity of auramine O，a dye for staining RNA and DNAare used to selectively gate platelet aggregates and platelet-neutrophil aggregates. Cells that fallin the gated area are checked for their images to correctly distinguish these aggregates from othercell components.Upon stimulation with ADP or serotonin，platelet-neutrophil aggregates was clearly detected with this new device. Now we are(2)我们examined the system which enables its quantitative measurementsphingolipid metabolism of peripheral blood cells, i.e., platelets, erythrocytes, neutrophils，mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in thesehighly-differentiated cells was their high sphingosine (Sph) kinase activity. About 40% of plateletSph 1-phosphate (Sph-1- p) could be released extracellularly by 12-O-tetradecanoylphorbol 13-acetatepossibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils，mononuclear cells，a significant percentage of Sph-1-P formed inside the cell was discharged without stimulation，while the stimulation-dependent release was marginal. Sph and its methylated derivative，N-dimethylsphingosine induced apoptosis not only in neutrophils but also in mononuclear cells，while Sph-l-P elicited Ca D22+ D2 mobilization in platelets. Our results suggest that all bloodcells may remove plasma Sph, which is harmful or suppressive to cellular functions，and change it into Sph-1-P, acting as the source of plasma Sph-1-Pwhich may play a variety of important roles in blood vessels。

(1) We have developed a new device which can sensitively detect the presence of platelet aggregates and platelet-neutrophil aggregates in whole blood. A pulse laser and a CCD camera are attached to a flow cytometer, which is an automated reticulocyte analyzer, R-3000 (Sysmex, Japan). Forward scatter intensity and fluorescence intensity of auramine O, a dye for staining RNA and DNA, are used to selectively gate platelet aggregates and platelet-neutrophil aggregates. Cells that fall in the gated area are checked for their images to correctly distinguish these aggregates from other cell components.Upon stimulation with ADP or serotonin, formation of platelet-neutrophil aggregates was clearly detected with this new device. Now we are trying to develop a system which enables its quantitative measurement.(2) We examined the sphingolipid metabolism of peripheral blood cells, i.e., platelets, erythrocytes, neutrophils, and mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in these highly-differentiated cells was their high sphingosine (Sph) kinase activity. About 40% of platelet Sph 1-phosphate (Sph-1-P) could be released extracellularly by 12-O-tetradecanoylphorbol 13-acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils, and mononuclear cells, a significant percentage of Sph-1-P formed inside the cell was discharged without stimulation, while the stimulation-dependent release was marginal. Sph and its methylated derivative, N,N-dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, while Sph-l-P elicited CaィイD22+ィエD2 mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph-1-P, acting as the source of plasma Sph-1-P, which may play a variety of important roles in blood vessels.