| Background and ObjectiveThymus is a central immune organ where T cells differentiate and mature.The common precursor of bone marrow-derived lymphocytes enters the thymus through blood vessels and lymphatic vessels.The mature T cells in the thymus are released into the peripheral immune organs through blood vessels and lymphatic vessels.Blood vessels and lymphatic vessels are the only two pathways for cells to immigrate and emigrate from the thymus.Therefore,both blood and lymphatic vascular endothelial cells play a vital role in this process.IFN-γ,a cytokine with multiple biological functions,regulates the expression of MHC molecules and adhesion molecules in endothelial cells.On the other hand,the emigration of thymocytes from the thymus is also highly correlated with the differentiation and maturation of cells,and the expression of CD62L and sphingosine 1-phosphate receptor 1(S1P1).The migration of S1P1-positive thymocytes is medated by the chemotaxis sphingosine 1-phosphate(S1P).S1P is mainly produced by red blood cells,and its concentration in blood and lymph is higher than that of lymphoid organs.The gradenet of S1P concentration is necessary for cell to egress from lymphoid organs.Recent studies found that S1P lyase(SPL),which express on thymic stromal cells,thymic medulla PVS,blood and lymphatic vascular endothelial cells,can catalyze the irreversible degradation of S1P to maintain the low concentration of S1P in thymus,which significantly gurantee the egress of mature thymocytes from thymus.Myasthenia gravis(MG)is an organ-specific autoimmune disease.Previous studies have reported abnormal proportions of thymus and peripheral T cell subsets in MG patients,and generate a germinal center composed of Tfh and B cells specific for the AChR antigen is formed in the thymus,at the same time,MG patients with abnormally high expression of various chemokines in the thymus,suggesting that there is abnormal lymphocyte emigration or immigration between the thymus and the periphery of MG patients.Our previous study showed that the expression of IL-6,TGFβ1,and IFN-γmRNA in thymic microenvironment of MG patients was significantly increased compared with the control group,and the number of S1P1~+and CD62L~+thymocytes in thymus of MG patients were increased.Most of these cells accumulate in or around the vasculature,suggesting that MG patients may have abnormal thymocyte output.To investigate the relationship between MG thymocyte abnormal output and abnormally high expression of cytokines IL6,TGFβ1,IFN-γin the thymus microenvironment.In this study,the expression and distribution of S1P1,CD62L,CD31 and LYVE1 in MG thymustissuewerefirstlyanalyzedbyimmunohistochemistryand immunofluorescence,among them,S1P1 and CD62L are key molecules that mediate the emigration of thymocyte,CD31 is the marker molecule of endothelial cells,and LYVE1 is the marker molecule for lymphatic endothelial cells.And then human umbilical vein endothelial cells(HUVEC)and human lymphatic endothelial cells(HLEC)were cultured,after stimulating with cytokines such as IL6,TGFβ1 and IFN-γ,then detecting the expression of adhesion molecules,chemokines and SPL in HUVEC and HLEC by using real-time fluorescent quantitative PCR(RT-qPCR)and Western Blot.We analyzed the mechanism of MG microenvironment regulating on blood and lymphatic vascular endothelial cells mediating thymocyte egress,which provided a scientific basis for further study of the mechanism of MG thymus abnormality and improvement of diagnosis and treatment.Methods(1)Study subjects:The experimental group was clinically diagnosed MG patients,AChRAb positive,thymectomy,thymic pathology type non-thymoma,typical follicular hyperplasia;control group was non-MG heart surgery patients.(2)Expression of MG thymocyte emigration related molecules:Taking MG patients and control thymus,performing paraffin embedding,making tissue sections,and analysis of the expression,distribution and correlation of S1P1,CD62L,CD31 and LYVE1 in thymus tissue by using immunohistochemistry and immunofluorescence.The expression and distribution of ICAM-1,VCAM-1 and SPL in thymic tissue were detected by immunohistochemistry.Quantitative analysis of immunohistochemistry:The immunohistochemical results of paraffin sections of thymus tissue were quantitatively analyzed using ImageJ software,and the cumulative optical density(IOD)was used to indicate the positive degree of histochemical results.(3)Effects of IL6/TGFβ1/IFN-γon the biological characteristics of HUVEC:HUVEC was cultured in vitro,IL-6/TGFβ1/IFN-γstimulated HUVEC and detected the expression of ICAM-1,VCAM-1,SPL,HLA-A,HLA-DR,CCL5,CCL19 and CCL21mRNA by RT-qPCR;Western Blot was used to detect the changes of ICAM-1,VCAM-1,SPL,HLA-A and HLA-DR protein expression levels.(4)Effects of IL6/TGFβ1/IFN-γon the biological characteristics of HLEC:Flow sorting of HLEC in thymic tissue of children with congenital heart disease.HLEC was cultured in vitro,IL-6/TGFβ1/IFN-γstimulated HLEC and detected the expression of ICAM-1,VCAM-1,SPL,HLA-A,HLA-DR,CCL5,CCL19 and CCL21 mRNA by RT-qPCR;Western Blot was used to detect the changes of ICAM-1,VCAM-1,SPL,HLA-A and HLA-DR protein expression levels.(5)Effects of intrathymic injection of IL6/TGFβ1/IFN-γon thymus tissue of C57BL/6 mice:IL6/TGFβ1/IFN-γwas injected into the thymus of C57BL/6 mice.The tissue structure and the expression of ICAM-1 in the thymus were detected by immunohistochemistry.(6)Statistical analysis of the experimental results:statistical analysis of the experimental results using SPSS 21.0 statistical analysis software,first to conduct a normality test,once in line with the normal distribution,using the t test between the two samples,using the mean±standard deviation to represent the results.It is indicated that when P<0.05,the difference is considered significant and statistically significant.Experimental result(1)Expression of MG thymocyte emigration related molecules(1)Expression of S1P1,CD62L,CD31 and LYVE1 in MG thymus:Immunohistochemistry and immunofluorescence showed that the corticomedullary structure of the thymic tissue was less distinct and the medulla was smaller in MG patients.S1P1~+thymocytes and CD62L~+thymocytes were increased,and they were mostly distributed in and around the vasculature in thymic tissue of MG patients.CD31~+endothelial cells and LYVE1~+lymphatic endothelial cells were increased,and were distributed in the cortex and medulla in thymic tissue of MG patients.(2)Expression of ICAM-1,VCAM-1 and SPL in MG thymus:The number of ICAM-1~+cells,VCAM-1~+cells and SPL~+cells in thymic tissue of MG patients were increased.ICAM-1~+cells and SPL~+cells were mostly distributed in the medullary region,and VCAM-1~+cells were mostly distributed in the lobule.In summary,it suggests that thymocyte egress increased in MG patients.(2)Effects of IL6/TGFβ1/IFN-γon the biological characteristics of HUVEC(1)Changes in adhesion molecules,chemokines and SPL mRNA levels in HUVEC:The levels of ICAM-1,SPL,HLA-A,HLA-DR and CCL5 mRNA were significantly increased after IFN-γstimulating HUVEC(P<0.05),VCAM-1 mRNA level was decreased early,but increased at 72 h,CCL19 and CCL21 mRNA levels were decreased(P<0.05);The levels of ICAM-1,VCAM-1 and SPL mRNA were increased after IL6 stimulating HUVEC(P<0.05),and the levels of HLA-A and HLA-DR mRNA were not significantly changed(P>0.05),the level of CCL21 mRNA was decreased early and then increased,CCL5 and CCL19 mRNA level were decreased(P<0.05);The expression of ICAM-1 and SPL mRNA in TGFβ1 stimulation group were increased early(P<0.05),the level of VCAM-1 mRNA was transiently increased,the level of HLA-A mRNA was decreased at 72 h(P<0.05),and HLA-DR mRNA level was decreased at 24 h(P<0.05),the mRNA levels of CCL5,CCL19 and CCL21 were significantly decreased(P<0.05).(2)HUVEC expresses changes in ICAM-1,SPL,HLA-A,and HLA-DR protein levels:The expression levels of ICAM-1,SPL,HLA-A and HLA-DR protein were increased after IFN-γstimulating HUVEC;The expression levels of ICAM-1and SPL protein were increased after IL6 stimulating HUVEC;The expression levels of ICAM-1 and SPL protein were increased after TGFβ1 stimulating HUVEC.(3)Effects of IL6/TGFβ1/IFN-γon the biological characteristics of HLEC(1)Changes in adhesion molecules,chemokines,and SPL mRNA levels in HLEC:ICAM-1,VCAM-1,SPL,HLA-A,HLA-DR,and CCL5 mRNA levels were significantly increased after IFN-γstimulating HLEC(P<0.05),CCL19 mRNA levels were reduced early and were increased at 72 h,CCL21 mRNA levels were significantly reduced(P<0.05);The level of ICAM-1,HLA-A,CCL19 and CCL21 mRNA were decreased early after IL6 stimulating HLEC,and the expression of SPL mRNA was not significantly different(P>0.05),the mRNA levels of VCAM-1,HLA-DR and CCL5 were increased with time,and were significantly increased at 72 h(P<0.05);The mRNA levels of ICAM-1,VCAM-1 and HLA-DR in TGFβ1-stimulated group were significantly decreased(P<0.05),and there was no significant difference in SPL mRNA expression(P>0.05),the mRNA levels of HLA-A,CCL5,CCL19 and CCL21were decreased early(P<0.05).(2)HLEC expresses changes in ICAM-1,VCAM1,SPL,HLA-A,and HLA-DR protein levels:The expression levels of ICAM-1,VCAM-1,SPL,HLA-A and HLA-DR protein were increased after IFN-γstimulating HLEC;the expression of HLA-DR protein was increased in IL-6-stimulated group;The level of HLA-DR protein expression was decreased in the TGFβ1-stimulated group.(4)Effects of intrathymic injection of IL6/TGFβ1/IFN-γon thymus tissue of C57BL/6 miceAfter intrathymic injection of IL6/TGFβ1/IFN-γin C57BL/6 mice,the histochemical results suggested that the thymic tissue of mice injected with IFN-γor IL6 showed that the boundary of the corticomedullary structure was not obvious,and the medulla area was reduced.After injecting IFN-γ,the expression of ICAM-1 in the thymus of mice increased significantly,and these changes were similar to those of MG patients.ConclusionThe abnormally high expression of IFN-γin the MG thymic microenvironment can promote egress of S1P1~+and CD62L~+cells from the thymus by increasing the expression of SPL and adhesion molecules on blood and lymphatic vascular endothelial cells. |
