Effects Of Platelet Upon The Adhesion Of Leucocyte With Liver Sinusoidal Endothelial Cell And Its Transmigration Through Endothelial Cells Undergoing Hypoxia-reoxygenation

Hepatic ischemia-reperfusion injury (IRI), which leads to common pathological damage during liver transplantation operation, is an important cause of liver dysfunction postoperatively. The occurring mechanisms of IRI are rather complicated, of which liver sinusoidal stasis and activation of neutrophils are important factors for reperfusion injury. During hepatic ischemia-reperfusion, the adhesions of platelet-liver sinusoidal endothelial cell (LSEC) and activated platelet-leucocytes increase markedly. However, it still remains unclear whether or not platelet plays a direct mediating role in the adhesion of leucocytes-LSEC. Further, the effects of adherent platelet and leucocytes upon the transmigration of leucocytes through LSEC have also remained poorly understood. This study is to explore the effects of platelet upon the adhesion of leucocyte-LSEC as well as the transmigration of leucocyte through LSEC, and to elucidate the relevant molecular mechanism during ischemia-reperfusion process, which would be of great significance for the further clarifying of the pathophysiology on the ischemia-reperfusion injury.Objective To investigate the effects of platelet on intercellular adhesion between leukocyte and liver sinusoidal endothelial cell and the transendothelial migration under the hypoxia-reoxygenation condition, as well as the underlying mechanism of the relevant adhesion molecules.Method Human liver sinusoidal endothelial cell (LSEC) was used for this study as well as leukocytes and platelets separated from healthy human peripheral blood. LSEC underwent the treatment of hypoxia-reoxygenation (H-R) for24and2hours respectively to mimic the process of ischemia-reperfusion injury (IRI). LSEC cultured under normal or H-R conditions was co-incubated with BCECF-labeled platelets. Fluorescence plate reader was employed to measure the fluorescence intensity of adherent platelets. LSEC was co-incubated with BCECF-labeled leukocytes and DiI-labeled platelets, which is cultured under normal or H-R conditions. Laser scanning confocal microscope was used to observe the adhesion of platelet-leukocyte. Leukocytes adherence to LSEC were investigated in which LSEC were cultured under normal or H-R condition and pre-incubated with or without platelets. Fluorescence plate reader was employed to measure the adhesion of leukocyte-LSEC. The experiment of antibody blockage was used to analyze the relevant cell adhesion molecules. The direct evidence of the role of platelets in mediating leukocyte-LSEC adhesion was derived by the laser scanning confocal microscope and transmission electron microscope. BCECF-labeled leukocytes were co-cultured with LSEC pre-incubated with platelets or not, which were inoculated in the transwells. Fluorescence plate reader was used to detect the number of migrated leukocytes.Results Adhesion of platelets to LSEC was increased significantly after H-R. The difference between resting and H-R group was statistically significant. Adhesion of platelets to leukocytes was also promoted obviously under H-R condition. Adherence of leukocytes on hypoxia-reoxygenated LSEC was significantly increased when LSEC was pre-incubated with platelets in comparing with the control group. The increase of leukocytes adherence correlates with the number of platelets positively, and the adhesion can be inhibited by monoclonal antibodies against GP I b, GP â…¡b, GPâ…¢a, P-selectin, CD31, ICAM-1, VCAM-1and ELAM-1, respectively. Furthermore, we got direct evidence that adhesion of leukocytes to LSEC was mediated by platelets through transmission electron microscope and laser scanning confocal microscope. The adhesion of platelets upon LSEC markedly reduced the transmigration of leucocytes through LSEC which is cultured under H-R condition.Conclusion The adhesion molecules of LSEC and leucocytes are present on the surfaces of platelets. Platelets firstly adhere to LSEC under H-R condition, and then the adherent platelets act as a bridge to mediate the adhesion of leucocytes-LSEC and inhibit the transmigration of leucocytes. This pathologic process accentuates the sequestration of leucocytes within liver sinusoid, and deteriorates liver ischemia-reperfusion injury.