| BackgroundNon-Hodgkin lymphoma(NHL),originating from lymphatic tissue,was the diversified immunocytoma.As the 7th place in the new cases of malignance,NHL accounted for 4%of them.According to the reports,3%of the patients who died for cancer or relative diseases,were due to NHL.Diffuse large B cell lymphoma(DLBCL)was the most common subtype of NHL,which was the typical heterogeneous disease.However.the etiologies and pathogenesis of DLBCL were still unclear.There were lots of non-tumor cells in DLBCL,including T cells subsets,macrophages,mast cells and so on.Not only DLBCL was the result of autonomous proliferation in tumor cells,but also its survival and proliferation depended on the signals coming from the microenvironment.Therefore,to investigate that how the inflammatory cytokines included in the DLBCL interact with tumor cell provided discovering the pathogenesis of DLVCL with new fields.From 2003,the relationship between inflammation and tumor gained more and more attentions,especially the interaction between the chemotactic factor and its receptors,as the focus of investigation on inducing inflammatory cytokines invasion.Basing on those mentioned above,we used GeneSifter online and BRB-ArrayTools to analyze the gene chip data of the DLBCL in the GEO(Gene Expression Omnibus),in order to find the cytokines and chemotactic factors closely related to lymphoma.The results showed that the novel chemokine CXCL16(chemokine ligand 16)and its receptor CXCR6(chemokine receptor CXC 6)were closely related to NHL.Whether CXC chemokine ligand 16(CXCL16)/CXC chemokine receptor 6(CXCR6)participating in the interaction between the inflammatory cytokines of DLBCL and tumor cells,and if it was,what kinds of inflammatory cytokines the interaction making biological effects on,there still needed further investigations to explain the relationship between inflammatory cytokines and DLBCL,in order to supply the prevention with more new ideas.1.CXCL16/CXCR6CXCL16 also named as scavenger receptor of phosphatidylserine(PS)and oxidized lipoprotein,belonged to CXC chemokine family,which was with the function as a receptor.Human CXCL16 gene located on 17p13 and encoded 254 amino acids(AA).There were two kinds of CXCL16,trans-membrane CXCL16(TM-CXCL16)and soluble CXCL16(sCXCL16).A disintegrin and metalloproteinase(ADAM)can promote the shedding of the extracellular domain of TM-CXCL16 to become sCXCL16.CXC chemokine receptor 6(CXCR6),whose gene located on the chromosome 3,was the only receptor of CXCL16,consisting of 7 transmembrane domain sequences with extracellular N-terminal and intracellular C-terminal.2.Current research of CXCL1 6/CXCR6There were many biological functions in CXCL16,with the mainly functions as the scavenger receptor and chemotactic factor.Recent researches mostly focused on the human diseases related to immune and inflammation.For example,CXCL16/CXCR6 could induce and activate the immunocytes including T cells,macrophages and so on,which could promote the pathogenesis and developments of atherosclerosis(AS),rheumatic arthritis(RA),uarthritis and disseminated sclerosis,as well as played an important role in the clinical progress of hepatitis,pneumonia and so on.Over the past few years,many scientists found that CXCL16/CXCR6 co-expressed in many kinds of tumor tissue and cells,such as breast cancer,pancreatic cancer,prostatic cancer and so on.This phenomenon indicated that CXCL16 was with the autocrine function in tumor cells.Not only the expression of CXCL16/CXCR6 in the variety kinds of tumors,but also they were with different influences in the prognosis.Previous researches showed that,CXCL16/CXCR6 could promote the metastasis and invasion of the cervical carcinoma,urinary bladder carcinoma,ovarian carcinoma,neuroblastoma and malignant melanoma,as well as participating in the malignant transformation of neuroblastoma and the differentiation of lymphoma.However,CXCL16/CXCR6 were also proved to inhibit the invasion and metastasis of gastric and renal cancers tumor,and improve the poor prognosis.Otherwise,there were some evidences indicated that serum sCXCL16 concentration could be the index as the diagnosis and prediction of liver metastasis,which indicated the potential value of sCXCL16 in clinical application.It was easy to conclude that CXCL16/CXCR6 over-expressed in many malignant tumors,but their functions were different for disease types.In the tumor of lymphoid and hematopoietic system,the studies on the CXCL16/CXCR6 got a late development.By searching on Pubmed database until February of 2016,there were 4 articles involving the over-expressing CXCL16/CXCR6 in lymphoma but not relating to the functions and mechanisms.In the early researches,Hanamote et al.were the first to find that in the expressing situation of chemotactic factors in a series of tumors related to inflammation,CXCL16 expressed to a certain level in the cancer cells from hodgkin lymphoma(HL).However,there were no more investigations on the significances of the CXCL16 expression.In the detection on lymphoma tissues of 39 cases,Darash-Yahana M et al.found that CXCL16 expressed highly in 25 cases(64.10%),but without subtype analysis.Deutsch et al.discovered that during the process of gastrointestinal mucosa-associated lymphoma tumors(MALT)transforming into DLBCL,the chemokine receptors including CXCR6 were up-regulated.In China,we were the first to focus on the potential functions of CXCL16/CXCR6 in lymphoma.By using quantitative-polymerase chain reaction(q-PCR),western blot(WB),confocal immunofluorescence(IF)methods and so on,We detected the expressions of CXCL16 and CXCR6 on mRNA and protein levels in different kinds of lymphoma cell lines,and they found that CXCL16 expressed widely in the lymphoma cell lines,including B-cell and T-cell lymphoma and HL.However,there were some differences among the different cell lines.In DLBCL cell line,CXCL16 and CXCR6 were found to co-express.By enzyme-linked immunosorbent assay(ELISA),DLBCL cell line could secrete sCXCL16.Although CXCL16/CXCR6 co-expression was observed in DLBCL cell lines,but expression in clinical specimens was not fully understood.The correlation between the expression of CXCL16/CXCR6 and clinical pathological features has not been reported.Therefore,the aim of this study was to observe the expression and clinical significance of CXCL16/CXCR6 in DLBCL tissues.In view of co-expresse of CXCL16/CXCR6DLBCL in tissues and cells,we can not help thinking:whether a high level of sCXCL16 means an autocrine effect?and how the mechanism?These become the second objective of this study.Therefore,in order to investigate on whether the sCXCL16 secreted by DLBCL was with autocrine function,we constructed the exogenously recombination protein sCXCL16 in the tumor cells,but the results showed that there were little effects of sCXCL16 on the proliferation of the tumor cells.However,in the combined applications with four cytokines,we found that sCXCL16 could work in coordination with tumor necrosis factor-α(TNF-α)and inhibit the proliferation of OCI-Ly8 and OCI-Ly10 cell lines,as well as promoting the apoptosis of them.What is the mechanism of the synergistic effect of TNF-α and sCXCL16 on the proliferation of DLBCL?This is the third goal of this study.TNF-α originated mainly from the M1 macrophage according to the reports.How macrophages chemotaxis to DLBCL tumor tissue is not clear yet.In NHL’s other type of follicular lymphoma,Monocytes can be recruited by CCL2 derived from the secretion of stromal cells be induced to promote the formation of tumor phenotype.This suggests that chemokines may play a role in the chemotaxis of inflammatory cells in the tumor.Then,this study aimed to investigate whether sCXCL16 is involved in the interaction between DLBCL cells and M1 macrophages that are often present in DLBCL.In view of the solution the disintegrin metalloproteinases(ADAM)in TM-CXCL16 to sCXCL16 transformation and the relationship between the ADAM and TNF-α,In the end of this study,we also discussed the relationship between the secretion regulation of sCXCL16 and the relationship with TNF-α in DLBCL.Objectives1.To investigate the expressions of CXCL16/CXCR6 in DLBCL tissues and cells and analyze the clinical significances;2.To investigate autocrine effect and mechanism of sCXCL16 in DLBCL tumor cells;3.Discussion on the mechanism of sCXCL16 involed in the interaction between tumor cells and M1 macrophages..Methods and Results1.Expression and clinical significances of CXCL16/CXCR6 in DLBCL tissues and cells1.1 Methods46 patients were diagnosed as DLBCL in Nanfang Hospital from 2009 to 2012.The data were collected from the medical records and the patients were followed up.Two tissue chips were obtained from Biomax(America),including 76 DLBCL cases.OCI-Ly3,OCI-Ly8 and OCI-Ly10 were chosen as the subjects.ICC,IF,RT-PCR and WB methods were used to detect the CXCR6 expression in the DLBCL cell lines.The CXCL16/CXCR6 proteins in the tissue chips and 46 tumor samples were detected by IHC.1.2 Results(1)The results of IHC detection showed that the expressing level of CXCL16 protein in DLBCL was higher than in the control(tissue chips 69.74%vs.control group 7.41%;tissue samples76.09%vs.control group7.41%),significantly(P<0.05).On the contrary,CXCR6 expression was lower than in the control(tissue samples 73.71%vs.control group 7.41%),significantly(P<0.05).(2)In the DLBCL tissue of the 46 cases,CXCL16 and CXCR6 co-expressed,which was with significant differences comparing to the control(65.22%vs.7.41%,P<0.05);CXCL16 expression was positively correlated to CXCR6(r=0.293,P=0.000),which indicated that DLBCL was with the potential autocrine function.(3)The CXCL16 expression in males(male vs.female,Z=-5.74,P=0.00)and in the patients below 60 years old was significantly higher(60y≤vs.>60y,Z=-7.24,P=0.00),as well as to the CXCR6 expression.(4)The average overall survival rates of the DLBCL patients with CXCR6+was lower than in those with CXCR6",significantly(P=0.007);the average overall survival rates of the DLBCL patients with CXCL16/CXCR6 co-expression was lower than in those without,significantly(P=0.008).The results of Cox’s proportional hazard model analysis showed that CXCL16/CXCR6 co-expression was the independent risk factor in the poor prognosis of DLBCL(RR=12.996,95%CI=1.727-97.377,P=0.013).2.Effects of sCXCL16 on the biological characteristics of DLBCL2.1 MethodsCCK-8 method was used for detecting the proliferation of DLBCL cell lines,Transwell method was for metastasis,and flow cytometry was for cell cycle and apoptosis.2.2 Results(1)There were no obvious effects of transfecting with exogenous sCXCL16 on the proliferation,cell cycle and apoptosis in DLBCL cell lines.(2)There were obvious effects of transfecting with exogenous sCXCL16 on the metastasis in DLBCL cell lines(OCI-Ly3:F=134.48,P=0.00;OCl-Ly8:F=39.59,P=0.00;OCI-Ly 10:F=85.43,P=0.00),which was with the dosage effects that as the sCXCL16 concentration increased,the metastatic ability of the DLBCL cell lines increased obviously.(3)4 common cytokines were combined with sCXCL16 and studied in the tumor microenvironment(TME),separately.The results showed that there were obvious inhibition of sCXCL16 combined with TNF-a or interferon-γ(IFN-γ)on the cell proliferation in OCI-Ly1,OCI-Ly3,OCI-Ly8 and OCI-Ly10 cell lines;anti-CXCR6 antibody could antagonize the inhibition partially;there was no obvious effect when sCXCL16 combined with interleukin-3(IL-3)or IL-6.(4)In OCI-Ly8 and OCI-Ly10 cell lines,there was no obvious effect of sCXCL16 combined with TNF-α on the cell cycle,whether in G1,S,G2 or M stage(P>0.05).(5)Comparing to the control,sCXCL16 combined with TNF-α could obviously induce the early apoptosis in OCI-Ly8 and OCI-Ly10 cell lines,while the anti-CXCR6 antibody could partially antagonize the induction;comparing to the control,sCXCL16 combined with TNF-α could induce the expression of apoptosis-pathways proteins(Capase3/Capase8/Bcl-xl),while the anti-CXCR6 antibody could partially antagonize the induction.3.Mechanism of TNF-α combined with sCXCL16 synergistic inhibit DLBCL proliferation3.1 MethodsThe gene expression difference between sCXCL16 and/or TNF-α was detected by gene chip screening in the DLBCL cells.ELISA was used to detect the effect of sCXCL16 on the release of TNF-α concentration in DLBCL cell lines;WB method was used to detect the sCXCL16 and the activation of NF-κb pathway;effect of PDTC antagonizing sCXCL16 on the release of TNF-α was detected by ELISA;WB method was used to verify the influence of sCXCL16 on TNFRSF12A expression;CCK-8 method was used to verify that the inhibitor PDTC and anti-TNFRSF12A antibody could antagonize the combined inhibition of sCXCL16+TNF-α on DLBCL cell proliferation.3.2 Results(1)Biological companies carried out the whole genome expression microarray screening.Differential expression of chemokine receptor pathway genes was found,including the receptor family TNFRSF12A gene of TNF-α.WB results verified that exogenous sCXCL16 could increase TNFRSF12A expression.(2)Exogenous sCXCL 16 could increase TNF-α concentration(OCI-Ly8:F=167.46,P=0.00;OCI-Ly10:F=94.49,P=0.00;);adding PDTC,as the inhibitor of NF-Kb could inhibit the effect partially.(3)The results of WB showed that in the sCXCL16 group,P-65(RelA),RelC,P105/P50 expressions increased in NF-κb pathway,while Rel-B expression did not changed obviously.(4)Adding PDTC and anti-TNFRSF12A antibody could antagonize the combined inhibition of sCXCL 16+TNF-α on DLBCL cell proliferation.4.sCXCL16 participates in the interaction between DLBCL cells and M1 macrophages4.1 MethodsDetection of M1 CXCR6 expression in tissue of DLBCL tissue by double staining IHC and IF.IHC was used to detect CD68 and CXCL16 expressions in 20 DLBCL tissues;adhesion method was used to separate the macrophages and induce M1 macrophage formation with LPS and IFN-y;tumor cells and M1 macrophages were co-culturing indirectly,in order to detect the indexes as following:CDCCK-8 method was used to detect the optical density(OD)value of the cells in the lower chamber;②ELISA method was used to detect the serum TNF-α and sCXCL 16 concentrations;③WB method was used to detect the expression apoptosis-pathway proteins of the tumor cells after co-culturing;④Transwell method was used to detect the chemotaxis of sCXCL 16 on M1 macrophages.4.2 Results(1)IHC and IF methods proved the CXCR6 expression in M1 macrophage of DLBCL tissue.(2)There was correlation between CXCL16 expression and cell number of CD68+in DLBCL tissues(r=0.813,P=0.000);there was chemotaxis of adding exogenous sCXCL16 in the lower chamber of Transwell to the M1 macrophages in the upper chamber,which was dose-dependent,showing as that the higher the sCXCL16 concentration,the more metastatic M1 macrophages.This suggested that sCXCL16 involded in the chemotaxis to M1 type macrophages.(3)M1 macrophages had inhibition on the proliferation of DLBCL cell lines by co-culturing M1 macrophages with OCI-Ly8 and OCI-Ly10,indirectly(vs.control group,OCI-Ly8:t=3.11,P=0.04;OCI-Ly10:t=16.86,P=0.00);sCXCL16 and TNF-α concentrations both increased in the co-culturing;the apoptosis proteins,Capase3/8 expression,increased after co-culturing,which indicated that co-culturing could increase TNF-α concentration and induce the apoptosis proteins expression and increase the cell apoptosis.5.Secretion regulation of sCXCL16 in DLBCL cells5.1 MethodsSerum sCXCL16 concentration was detected by ELISA;ADAM 10 expression in DLBCL cell lines was detected by WB;And analyze the relationship between them.Effects of exogenous on the concentration of sCXCL16 in the culture supernatant of DLBCL cell line.5.2 Results(1)There were differences between the secretion of sCXCL16 in the 4 DLBCL cell lines,significantly(F=24.706,P=0.000);ADAM 10 expressions were found in both DLBCL cell lines and the gray value of them were detected by Image J software;the results showed that there was high correlation of ADAM 10 expression and sCXCL 16 concentration(r=0.89,P=0.04),appearing as the stronger ADAM 10,the higher the sCXCL 16 secretion.(2)Adding IFN-γ or TNF-α could obviously increase serum sCXCL16 concentration,while there was no obvious effect with adding IL-3 or IL-6;the inhibitor of ADAM 10 could antagonize the effect of TNF-α on the sCXCL16 secretion from DLBCL.TNF-α could increase the ADAM 10 expression,which indicated that TNF-a could promote the serum sCXCL16 concentration increasing by inducing ADAM 10 expressionConclusions1.CXCL16 and its receptor CXCR6 expressed widely in DLBCL tissues and cell lines;CXCL16/CXCR6 co-expression was the independent risk factor in the poor prognosis of DLBCL;2.The experiments in vitro showed that there was no obvious effect of sCXCL16 on the cell cycle,apoptosis and proliferation in DLBCL cell lines,but sCXCL16 could induce the metastasis of tumor cells;sCXCL16 combined with TNF-α could promote the cell apoptosis;3.On autocrine mechanism of sCXCL16 in DLBCL cells,we found that sCXCL16 can induce the expression of TNF-a receptor family TNFRSF12A in DLBCL cells,which played the role of synergistic inhibition of DLBCL proliferation and promoted the apoptosis of tumor cells with TNF-α.A certain concentration of sCXCL16 can also induce DLBCL tumor cells to produce TNF-α,which involves the activation of NF-kappa B pathway.4.M1 type macrophages express the receptor CXCR6 on the cell surface in DLBCL tissue.Soluble CXCL16 can chemotaxis M1 type macrophage.The interaction of M1 type macrophages to DLBCL includes:1)Inhibition of DLBCL cell proliferation;2)To promote the secretion of TNF-α;3)To activate DLBCL cell apoptosis pathway and induce cell apoptosis.5.There is a positive correlation between the concentration of sCXCL16 in the supernatant medium and the expression level of ADAM 10 in DLBCL cells.TNF-αin tumor microenvironment can regulat the sCXCL16 concentration by induce ADAM 10 expression in DLBCL tumor cells.Innovations:1.The first time to explain the clinical significances of CXC16/CXCR6 in DLBCL and verify the influences of sCXCL16 on the biological function of DLBCL.2.Observation of autocrine effect of sCXCL16 in DLBCL cells in vitro.The synergistic inhibitory effect of sCXCL16 combined with TNF-α on the proliferation of DLBCL was found.Discovery of the secretion of TNF-α in the autocrine way which is induced by sCXCL16.By inducing the expression of TNF-α,ADAM 10 can increase the concentration of sCXCL16 in the supernatant,which made the autocrine loop effect.sCXCL16 up-regulats the receptor TNFRSF12A expression.The discovery of the TNFRSF12A receptor provides an experimental basis for exploring the possible target of the clinical treatment of DLBCL.3.Whether sCXCL16 is involved in the interaction between DLBCL tumor cells and M1 macrophages and the regulation of sCXCL16 secretion by ADAM 10 was in a more systematic study.It was proposed and validated that the tumor cells secrete sCXCL16 and combined with CXCR6 of macrophages,which maybe urges M1 to secrete TNF-a,and then produce the side effects cycle. |
PDF Full Text Request