Pathogenetic Role Of Tissue Factor And Proteinase-activated Receptor In Immune-mediated Endothelial Injury During Acute Graft-versus-host Disease

PartⅠGene and Protein Expression of Proteinase-activatedReceptor-1, 2 in a Murine Model of Acute Graft-versus-host DiseaseObjective: The mechanism of acute GVHD was known without certainty, but theactivation of donor T cells and release of the proinflammatory cytokines such as TNF-α,IFN-γand IL-1 may be involved.Recent findings have demonstrated PARs are not onlydegradative enzymes, but also important signaling molecules involved in inflammation andimmunology.The aim of this study was to explore the expression of proteinase-activatedreceptor-1, 2(PAR-1, 2) in a murine model of acute GVHD.Method: A murine model of acute GVHD after allogeneic HSCT was established, andsyngeneic HSCT mice were used as the controls.Quantitative real-time PCR, Western blotand immunohistochemical test were done to detect the gene and protein expression ofPAR-1, 2 in multiple organs of allogeneic HSCT mice and the controls.Result: Allogeneic HSCT mice showed classical symptoms and histological changesof acute GVHD.PAR-1 mRNA expression was significantly increased in the skin, liver,small intestine of allogeneic HSCT mice (skin: 0.039±0.013 vs.controls: 0.008±0.002,P=0.009; liver: 0.165±0.006, vs.controls: 0.017±0.006, P=0.004; small intestine:0.215±0.009 vs.controls: 0.016±0.002, P=0.003) but not in the stomach, lung and kidneyof allogeneic HSCT mice (P>0.05).PAR-2 mRNA expression in the liver and smallintestine of allogeneic HSCT mice (liver: 0.010±0.002 vs.controls: 0.003±0.001, P=0.008;small intestine: 0.006±0.001 vs.controls: 0.003±0.001, P=0.024) but not in the other organs(P>0.05) was found to be significantly elevated.The protein expression of PAR-1, 2 werein accordance with the mRNA expression as shown by western blot.The levels of PAR-1protein expression in the skin, small intestine, liver of allogeneic HSCT mice wereincreased by 5.3, 2.9 and 5.2-fold compared with the controls respectively (P<0.01).Thelevels of PAR-2 protein expression in the small intestine and liver of allogeneic HSCT micewere increased by 2.85 and 4.0-fold compared with the controls respectively (P<0.01). Immunohistochemical test showed there was strong PAR-1 and PAR-2 immunoreactivity inthe hepatocyte, vascular endothelial cell of the liver, epithelium mucosae, glandularepithelium of small intestine of allogeneic HSCT mice.Enhanced immunoreactivity ofPAR-1 was also found in epithelial cell and glandular epithelium of the skin.But there wasabsent or weak PAR-1 and PAR-2 immunoreactivity in the organs of controls and the lung,kidney, stomach of allogeneic HSCT mice.Conclusion: Increased expression of PAR-1, 2 in the target organs of acute GVHDsuggests PAR-1, 2 may contribute to pathogenesis of acute GVHD after allogeneic HSCT.It may lead to a better understanding of acute GVHD and may provide a promising methodto alleviate acute GVHDPartⅡProteinase-Activated Receptor-1 Mediates Allogeneic CD8~+ TCell-Induced Apoptosis of Vascular Endothelial CellsObjective:Vascular endothelial-cells injury plays a pivotal role in the pathogenesis ofgraft-versus-host disease and transplant-associated endothelial injury syndrome.In thiscontext, allogeneic reactive cytotoxic T cell may contribute to the apoptosis of vascularendothelial cells.The aim of this study was to investigate the molecular mechanism ofallogeneic CD8~+T cell-induced apoptosis of vascular endothelial cells.Method: Allogeneic CD8~+T cells were isolated from PBMC by positive selectionusing magnetic beads coated with anti-CD8 antibody.Apoptosis of human umbilical veinendothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs)were detected by AnnexinⅤ-FITC labeling.Gene and protein expression ofproteinase-activated receptor-1(PAR-1) in vascular endothelial cells were tested byRT-PCR and Western blotting.Western blotting was also used to detect the change ofMAPK and Caspase-3 expression in the vascular endothelial cells.The influence ofSFLLRN (PAR-1 agonist), ATAP2 (PAR-1 antibody), SB203580 (inhibitor of p38MAPK),SP600125 (inhibitor of JNK) on apoptosis were also examined. Result: After co-culturing with allogeneic CD8~+T cells for 24 h and 48 h, theapoptosis rates of HUVECs were 51.7±4.1% and 29.4±3.3% respectively (P<0.01, vsuntreated HUVECs), the apoptosis rates of HDMECs were 28.9±2.2% and 15.2±1.8%respectively (P<0.01, vs untreated HDMECs).The effect of PAR-1 agonist on apoptosis ofHUVECs and HDMECs was similar to the effect of allogeneic CD8~+T cells.These effectswere largely prevented by ATAP2 and SB203580 (p<0.05).ATAP2 reduced allogeneicCD8~+T cells-induced apoptosis of HUVECs and HDMECs by 82.8%, 76.5% comparedwith the untreated cells respectively (P<0.05).SB203580 reduced allogeneic CD8~+Tcells-induced apoptosis of HUVECs and HDMECs by 76.3%, 81.6% compared with theuntreated cells respectively (P<0.05).Allogeneic CD8~+T cells and PAR-1 agonist enhancedcleavage of Caspase-3 and led to p38MAPK phosphorylation.The levels of Caspase-3protein expression in HUVECs and HDMECs were increased by 4.7, 1.9-fold comparedwith the controls respectively at 12-h point (P<0.01).The levels of phosphorylatedp38MAPK in HUVECs and HDMECs were increased by 8.8, 5.3-fold compared with thecontrols respectively (P<0.05).Conclusion: Allogeneic CD8~+T cells induced apoptosis of vascular endothelial cellsthrough PAR-1 dependent modulation of intrinsic apoptotic pathway via cleavage ofCaspase-3 and phosphorylation of p38MAPK.It may lead to a better understanding ofacute GVHD and transplant-associated vascular disease.Measures to block PAR-1 mayalleviate acute GVHD and transplant-associated endothelial injury syndrome.PartⅢExpression of Tissue Factor in a Murine Model of AcuteGraft-versus-host DiseaseObjective: The aim of this study was to explore the expression of tissue factor (TF) ina murine model of acute graft-versus-host disease (GVHD).Method: A murine model of acute GVHD after allogeneic HSCT was established, and syngeneic HSCT mice were used as the controls.Quantitative real-time PCR and Westernblot test were done to detect the gene and protein expression of TF in multiple organs ofallogeneic HSCT mice and the controls.Result: Allogeneic HSCT mice showed classical symptoms and histological changesof acute GVHD.The levels of TF mRNA expression in the skin, stomach, small intestine,liver of allogeneic HSCT mice were increased by 15.1±2.1, 5.5±1.4, 9.7±2.3, 14.3±2.9-foldcompared with the controls respectively (P<0.01).The levels of TF protein in the skin,stomach, small intestine, liver of allogeneic HSCT mice were increased by 13.5±2.7,6.2±0.9, 7.9±1.6, 15.3±3.2-fold compared with the controls respectively (P<0.01).ButmRNA and protein expression of TF in the kidney and lung of allogeneic HSCT mice wasnot elevated compared with the controls.Conclusion: Increased expression of TF in the target organs of acute GVHD suggestsTF may contribute to pathogenesis of acute GVHD after allogeneic HSCT.It may lead to abetter understanding of acute GVHD and may provide a promising method to alleviateacute GVHDPartⅣPathogenetic Role of Tissue Factor in acuteGraft-versus-host DiseaseObjective: Recent findings suggest that tissue factor (TF) play an important role intransplant immunology.The aim of this study was to study the pathogenetic role ofallogeneic T lymphocytes and tissue factor (TF) in GVHD.Method: TF mRNA and protein expression in organs of allogeneic HSCT mice andthe controls were determined by real-time PCR and Western blot.The effect of allogeneic Tcells on TF, VCAM-1, TNF-α, IFN-γand IL-6 expression and activation of MAPKs inHUVECs was detected by flowcytometry, real-time PCR or Western blot.The influence ofTF antibody, SB203580 and SP600125 on allogeneic T cells-induced proinflammatory cytokines expression was also investigated.Result: 1.Allogeneic CD4~+T cell and CD8~+T cell enhanced TF, VCAM-1, TNF-α,IFN-γand IL-6 expression in TNF-αprestimulated HUVECs compared to the controls.2.Allogeneic CD4~+T cell and CD8~+T cell enhanced p38MAPK and JNK phosphorylation inHUVECs compared to the controls, but ERK phosphorylation was not affected.SB203580and SP600125 reduced allogeneic CD4~+T cell and CD8~+T cells -induced TF expression inHUVECs compared to non-treated HUVECs.3.TF antibody, SB203580 and SP600125down-regulated allogeneic T cells-induced VCAM-1, TNF-α, IFN-γ, IL-6 expression inHUVECs compared to non-treated HUVECs.Conclusion: TF is the key factor in immune-mediated endothelial-injury in acuteGVHD.Allogeneic T cells-induced expression of TF and other proinflammatory cytokinesin vascular endothelial cells may contribute to the pathogenesis of GVHD.Measures whichblock TF expression of vascular endothelial cells may inhibit production ofproinflammatory cytokines contributing to acute GVHD and alleviate acute GVHD.