ABSTRACT
Objective To study the role of endothelial cells on the inflammatory cytokine release in septic shock through the septic shock serum stimulating human primary endothelial cells (HPAEC) and peripheral blood mononuclear cells(PBMC).Methods PBMC isolated from healthy people by density gradient centrifugation.HPAEC cell surface markers CD144 and von Willebrand factor(vWF) molecule expression by RT-PCR and Western blot.Serum levels of IL-6,TNF-α,MCP-1 from septic shock patients and healthy human detected by ELISA.HPAEC and PBMC were stimulated with the isolated serums and LPS,respectively.ELISA was used to detect the supernatant IL-6,TNF-α,MCP-1 levels.HPAEC membrane molecules ICAM-1 expression was detected by flow cytometry with serum shock and LPS stimulation.Supernatant levels of IL-6,TNF-α,MCP-1 of HPAEC with S1P1 receptor agonist CYM-5442 pretreatment was detected by ELISA after shock serum stimulation.Results Endothelial cell markers CD144 and vWF molecules could be detected in the HPAEC.Levels of inflammatory cytokines IL-6,TNF-α,MCP-1 in patients with septic shock serum were significantly higher than healthy people (P＜0.01).PBMC and HPAEC with LPS or shock serum treatment respectively,compared with normal group,levels of inflammatory cytokines in the culture supernatant were significantly higher(P＜0.01).For PBMC,the level of inflammatory cytokines between shock group and LPS group were not significantly different (P＞0.05).But for HPAEC,levels of inflammatory cytokines in the supernatant of the shock group compared to the LPS group was significantly higher (P＜0.01).Similarly,when two cells after LPS stimulation,IL-6,TNF-α levels of HPAEC's supernatant were significantly lower than PBMC' s (P＜0.01),MCP-1 levels was no difference (P＞ 0.05).But when the stimulation of shock serum,HPAEC of IL-6,TNF-α levels and PBMC no significant difference (P ＞0.05).MCP-1 was significantly increased (P＜0.01).Shock patients serum stimulation S1P1 receptorspecific agonist CYM-5442 pretreatment of HPAEC with pretreatment of S1P1 receptor specific agonist CYM-5442,the culture supernatant of inflammatory cytokines IL-6,TNF-α,MCP-1 levels were significantly lower (P＜0.01).Conclusion Endothelial cells may play a central role on the release of inflammatory cytokine during septic shock.
ABSTRACT
Objective To investigate the influence of extracellular high mobility group box 1 (HMGB1) on viral replication in HTLV-1 infected T cells.Methods HMGB1 in culture supernatants of adult T-cell leukemia virus 1 (HTLV-1) virus-negative cell:Jurkat,MOLT4 cells and HTLV-1 virus-positive cells:MT2,MT4,was detected by ELISA;The HTLV-1 long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into MT2 cells by Tfx-50-mediated transfection,and 0.25,0.50,0.75 μg/ml of HMGB1 polyclonal antibody(HMGB1 PcAb) and its isotype control rabbit IgG antibodies,0.03,0.1,0.3 μg/ml rhHMGB1 and its control PBS,were added into culture supernatant respectively,then luciferase activity was detected after 48 h;Similarly,0.25 μg/ml HMGB1 PcAb and the isotype control antibody,0.3 μg/ml rhH-MGB1 and the control PBS were added to the culture supernatant of MT2 cell,the viral gene,pol1,pol2,gag,env,etc,were performed by real-time PCR.Results Culture supernatant HMGB1 levels has no significant difference between HTLV-1 positive cells MT2 and MT4 and the other two virus-negative T cell lines;Compared with isotype control antibody group,the culture supernatant,to which is added 0.25 μg/ml HMGB1 PcAb,can significantly inhibit the HTLV-1-LTR transcriptional activity and suppress the expressions of the viral gene pol1,pol2,gag,env.Compared with the control PBS,0.3 μg/ml rhHMGB1 significantly promotes the transcriptional activity of the HTLV-1-LTR and the expressions of the viral gene pol1,pol2,gag,env.Conclusion The extracellular HMGB1 can promote viral replication of HTLV-1 infected T cells.