| BackgroundHodgkin lymphoma (HL) represents one of the most common subtypes of lymphoid system malignancies in the Western population, which accounts for approximately 11% of all malignant lymphomas. The incidence of HL is also rising in China in recent years. Clinically, most patients can be cured with early diagnosis and standard treatment strategies (chemotherapy, radiotherapy, and/or surgery), although approximately 20% will die after relapse or progressive disease. Histologically, HL is subdivided into Classical Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). Classical Hodgkin lymphoma, with its subtypes of nodular sclesis, mixed cellularity, lymphocyte rich and lymphocyte depleted, accounts for approximately 95% of all HL cases. HL is unique among all cancers because the malignant cells of Hodgkin lymphoma known as Hodgkin and Reed-Sternberg (HRS) cells usually make up only approximately 1% of the tumor tissue and reside in a complex admixture of cells with an inflammatory background, including T lymphocytes, B lymphocytes, plasm cells, eosinophils, neutrophils, histocytes, monocytes and fibroblasts. Therefore, not only HRS cells but also its microenviroment should be noticed in the research of Hodgkin lymphoma, including the background immune cells and the cytokines/chemokines.Tumor microenvironment formation is very essential for HL. HRS cells secrete many kinds of cytokines/chemokines which can play a wide variety of functions that work either in an autocrine manner to bind to the surface receptor of HRS cells themselves or in a paracrine manner to attract and modulate the surrounding background cells. The non-neoplastic reactive inflammatory cells also secrete many kinds of cytokines/chemokies to act on HRS cells. The interaction and crosstalk between HRS cells and non-neoplastic reactive inflammatory cells conjointly construct a complex molecular network, and contribute to the survival, proliferation, antiapoptosis, migration of HRS cells and evade host immune attack. The most abundant cells found in HL are infiltrating CD4+ T lymphocytes and the role of T regulatory (Treg) cells as one of main subsets is eye-catching. Treg cells have immunosuppressive potential as they can suppress the activation of cytotoxic lymphocyte (CTL), and assist to accomplish the immune escape of HRS cells. HRS cells can secrete some cytokines/chemokines to attract Treg cells and maintain their survival and activation.To explore HL microenvironment formation and the role of related cytokines/ chemokines, we used online software GeneSifter and BRB-Array Tools to analyze the gene expression differences between HRS cells separated from HL tumor tissues, CHL cell line (L428), Burkitt’s lymphoma cell line (Raji) and diffuse large B-cell lymphoma (DLBCL) cell from tumor tissues, and tried to find the closely related cytokine/chemokines with the HRS cells. The results showed that except RANTES/CCL5, CCL17, CCL22, SISd, IL-6, which have been illustrated already, new-found chemokine CXCL16 and its receptor CXCR6 are also essential for CHL. The differences of CXCL16/CXCR6 expression between CHL and DLBCL were further compared and we found that the expressions of CXCL16/CXCR6 were higher in CHL than in DLBCL. Furthermore, CXCL16 expression was markedly downregulated in houman CHL-like mouse model constructed by gene silencing technology. The role and expression status of CXCL16 and its receptor CXCR6 in HL caused our study attraction.CXCL16 is a recently discovered chemokine, as a member of the CXC chemokine subfamily, with two existing forms:transmembrane CXCL16 (TM-CXCL16) and soluble CXCL16 (sCXCL16). CXCL16 selectively express on antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, and its receptor CXCR6 express on naive CD8+T cells, natural killer T (NKT) cells, and type 1-polarized, activated CD4+ and CD8+ T cells. CXCL16/CXCR6 are involved in various physiological processes, such as cellular immunologic response, cellular adhesion, antigen presenting, inflammatory reaction and T cell development in thymus. Early studies of CXCL16 focused on the inflammation and immune-related disorders. It has been reported that CXCL16 plays important roles in migration of leukocytes of human inflammatory diseases including atherosclerosis, rheumatoid arthritis, hepatitis, pneumonia, nephritis and HIV infection disease. Recently, the functions of CXCL16 and its receptor CXCR6 on tumors are receiving increasing attention. It was reported that CXCL16/CXCR6 over-express in many malignancies, such as colorectal cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer and renal cell cancer. High expression of CXCL16 could stimulate growth, metastasis and recurrence in most reported tumors, while it may also play an opposite role in some others. Moreover, CXCL16/CXCR6 were involved in the formation of tumor microenvironment and stimulate anti-tumor immunity by inducing immune cells, such as CD4+/CD8+ T lymphocytes, NK lymphocytes and so on.As a chemokine axis, althoug CXCL16 and its receptor CXCR6 commonly express in immune cells like T lymphocytes, B lymphocytes, NK cells and macrophages, there were still seldom studies of CXCL16 on lymphatic hematopoietic system malignancies. Hanamoto firstly noticed that CXCL16 express in HL origin cell lines when they researched the chemokines related to Hodgkin lymphoma, but they hadn’t explored the significance of CXCL16 expression. Darash-Yahana detected the expression of CXCL16/CXCCR6 in a series of inflammation-associated cancers, including 28 cases of lymphoma which 25% cases highly expressed CXCL16, but they hadn’t furtherly mentioned the histological kinds and other characteristics of these reported lymphomas. Deutsch firstly noticed the role of CXCR6 on lymphoma malignant transformation when they researched chemokine receptors associated with progression in mucosa-associated lymphoid tumor (MALT) transformate to DLBCLTo compare the differences of CXCL16/CXCR6 expression in different lymphoma types, the expression of CXCL16/CXCR6 in 9 lymphoma cell lines (7 B cell origin,2 T cell origin) were detected by immunohistochemistry, real time quantitative PCR (RT q-PCR), western blotting, confocal microscopy and enzyme-linked immunosorbent assay (ELISA) in our previous study. The resullts showed that CXCL16 widly express in lymphoma cell lines with different expression levels in different origin lymphoma cell lines. The expression of CXCL16 was higher in Hodgkin lymphoma cell line L428 and some non-Hodgkin lymphoma cell lines, such as RPMI 8226, KM3 and Jurkat. As we all known, cell lines are simple cell model cultured in vitro, lacking of microenvironment and the influence of nutrition and host immune, so there are differences between cell lines in vitro and tumor tissues in vivo. But the expression and clinical significance of CXCL16 in lymphoma, especially in HL tissues, and the role of CXCL16 to HRS cell and its background inflammatory cells are still need to explored.Because the development of HRS cells is closely correlated to the abnormal activation of NF-κB signal pathway and lack of CD99 gene, the expression of CXCL16 and CD99 in HL and NF-κB signal pathway were observed in our previous research. It was showed that there is positive correlation between NF-κB signal activation and CXCL16 expression while negative correlation between CD99 expression and CXCL16 expression. Changing NF-κB activity can regulate CXCL16 expression in L428 cells. Phosphatidylinositol 3-kinase (PI3K)/AKT signal is another important signal pathway for HL. Constitutive activation of PI3K contributes to the survival of HL cells through a mechanism involving Akt kinase and mTOR signals. It was reported that CXCL16 signals via Gi, PI3K, AKT, I kappa B kinase, and nuclear factor-kappa B and induces cell-cell adhesion and aortic smooth muscle cell proliferation. In tumor, such as prostate cancer and hepatic cancer, high expression of CXCL16 can also via PI3K/Akt signal pathway and promote the clinical progress. In HL, how is the relationship between expression of CXCL16 and activity of PI3K/AKT/mTOR and NF-κB signal pathway?Thus, the exact expression status of CXCL16 in lymphoma, especially the role and its molecular mechanism of CXCL16 in HL, are the main objectives in this study.Objectives1. To detect the ecaxt expression status of CXCL16 and analysis its significance in lymphoma tissues, especially in HL, consummate and extand our previous researches;2. To explore the influence of CXCL16 expression to the proliferation ability, migration ability, cell cycle, apoptosis and immunophenotype of HRS cells, investigate the role of CXCL16 in the progression of HL;3. To explore the influence of CXCL16 expression to chemotaxis ability and proliferation ability of Treg cells in vitro, obsearve the interaction between L428 cells and Treg cells under co-cultured, investigate the role of CXCL16 in microenvironment formation of HL;4. To obsearve the activity of PI3K/AKT/mTOR and NF-κB signals in HL tissues and cell line and explore their roles to proliferation and migration ability of HRS cells, analysis the influence of CXCL16 expression to these two signals, approach the molecular mechanism of CXCL16 in HL.Methods1. Expression of CXCL16/CXCR6 were detected in lymphoma tissues and cell linesTo consummate and extand our previous researches, we firstly selected 70 lymphoma cases (including 45 HL cases) and detected the expression status of CXCL16 and its reportor CXCR6 proteins in these tissues by immunohistochemistry method, and then analysised the clinical significance based on the clinicopathological characteristics. The CXCL16/CXCR6 proteins expression were also detected by immunocytochemistry in lymphoma cell lines. Then optical density (OD) value of positive expression cells were analyzed by IPP6.0 software and the results were compared to which in lymphoma tissues.2. The role of CXCL16 to HRS cellsTo explore the role of CXCL16 to HRS cells, we firstly established CXCL16 over-expressed L428 cell subline by lentivirus transfection and exogenously applied recombinanted soluble CXCL16 (sCXCL16) and CXCR6 antibody (anti-CXCR6) protein to up-regulate or block the expression and secretion of CXCL16. Then the changes of biological characteristics, such as proliferation ability, migration ability, cell cycle, apoptosis and immunophenotype were measured by CCK-8 test, Transwell invasion assay, PI-Annexin V apoptosis assay and flow cytometry.3. The role of CXCL16 to HL microenvironment formationTo explore the influence of CXCL16 expression on HL microenvironment formation, we firstly sorted CD4+CD25+ Treg cells from healthy human peripheral blood mononuclear cells (PBMC) using immunomagnetic beads. Chemotaxis ability of CXCL16 to Treg cells was detected by Transwell assay. And then we obsearved cell morphology changes and detected the interactions of proliferation and migration ability of L428 cells and Treg cells by CCK-8 test in co-culture condition.4. The molecular mechanism of CXCL16 in HLFirstly, phosphorylation levels of the key proteins such as AKT, P70S6 and IκB of PI3K/AKT/mTOR and NF-κB signal pathways in HL tissues and cell lines were detected by immunohistochemistry, immunocytochemistry and western blotting. Secendly, to analysis the influence of CXCL16 on activities of these two signals, phosphorylation levels of the key proteins were detected again on different CXCL16 regulation levels. Finally, proliferation and migration ability of L428 cells were detected by Transwell assay and CCK-8 test after singlely adding PI3K signal inhibitor LY294002 and conbinatedly adding LY294002 and sCXCL16.Results1. Expression of CXCL16/CXCR6 in lymphoma tissues and cell lines(1) In lymphoma tissues, expression of CXCL16 was higher in CHL than in NHL (Z=-2.550, P=0.011); and in CHL there was positive correlation between the expression of CXCL16 and tumor size, while no correlation btween the expression of CXCL16 and patients gender (Z=-0.038, P=0.970), age (Hc=0.092, P=0.955) and tumor kinds (Hc=1.802, P=0.614);(2) It was showed that there are no significant differences between the expression of CXCR6 in CHL and NHL in total lymphoma cases (Z=-1.850, P=0.064); and in CHL there are no correlation between the expression of CXCL16 and tumor size(Z=-1.841, P=0.066), gender (Z=-0.275, P=0.784), age (Hc=0.894, P=0.640) of patients, or tumor kinds (Hc= 1.114, P=0.774);(3) There were statistical significances between the expression of CXCL16 in each lymphoma cell lines (P<0.05), in L428 it was the highest and then was Karpass299, Raji and Ly10 in turn; and the expression of CXCR6 in each lymphoma cell lines also had statistical significances (P<0.05), in L428 it was the lowest and then was Raji, Ly10 and Karpass299 in turn;(4) There was positive correlation between the expressions of CXCL16 and CXCR6 in lymphoma tissues (r=0.411, P=0.000), while negative correlation in lymphoma cell lines (r=-0.764, P=0.000).2. The role of CXCL16 to HRS cells(1) Construction and identification of over-expressed CXCL16 L428 cell subline by lentivirus transfectionCXCL16 overexpreesed lentiviral vector was constructed and then stably transfected L428 cells. Cell morphology was obsearved after transfection. Success of transfection was suggested with green fluorescence protein (GFP) obsearved under fluorescence microscope; results of Quantitative PCR test showed that CXCL16 mRNA is up-regulated in transfected L428 cell subline; results of Westrn blot assay showed that CXCL16 protien is up-regulated in transfected L428 cell subline; results of ELISA assay showed that excretory soluble CXCL16 protien increased in transfected L428 cell subline;(2) Cell proliferation ability of L428 in different CXCL16 expression levelTheresults of CCK-8 test showed that except the 1 st day, the cell proliferation abilities were showed statistical significances between different treated groups in other days (P<0.05). Endogenously over-expressed CXCL16 and ectogenesisly adding sCXCL16 can both effectively promote L428 cell proliferation (P<0.05), but there was no significant difference between this two treated measures; while ectogenesisly adding CXCR6 antibody can conversely decrease L428 cell proliferation;(3) Cell migration ability of L428 in different CXCL16 expression levelThe results of Transwell test showed that endogenously over-expressed CXCL16 and ectogenesisly adding sCXCL16 can both effectively promote L428 cell migration ability (P<0.05), but there was no significant difference between this two treated measures (P>0.05); while ectogenesisly adding CXCR6 antibody can conversely decrease L428 cell proliferation ability (P<0.05); morphology observation confirmedd that the big L428 cells may be transformated from medium size cells;(4) Cell cycle of L428 in different CXCL16 expression levelThe results of cell cycles which detected by flow cytometry showed there were statistical significances between untreated L428 cells and other different treated groups (P<0.05). It was showed that endogenously over-expressed CXCL16 and ectogenesisly adding sCXCL16 can both effectively decreased the percentage of cells in G1 phase and respectively increased the proportion of cells at S and G2/M phases (P<0.05), but there was no significant difference between this two treated measures (P>0.05); while ectogenesisly adding CXCR6 antibody can conversely increase the proportion of cells at G1 phase and decreased the percentage of cells in S and G2/M phases (P<0.05);(5) Cell apoptosis of L428 in different CXCL16 expression levelThe percentage of cell apoptosis which detected by flow cytometry were 5.69± 0.32,2.44±0.41,2.37±0.37 andl 1.62±0.83 in untreated L428, CXCL16+, sCXCL16 and anti-CXCR6 group, respectively. The results showed endogenously over-expressed CXCL16 and ectogenesisly adding sCXCL16 can both effectively decreased the percentage of cells in G1 phase and respectively increased the proportion of cells at S and G2/M phases (P<0.05), but there was no significant difference between these two treated measures (P>0.05); while ectogenesisly adding CXCR6 antibody can conversely increase the proportion of cells at G1 phase and decreased the percentage of cells in S and G2/M phases (P<0.05);(6) Immunophenotype change in different CXCL16 expression levelThe results of L428 immunophenotype detected by flow cytometry showed there were no no significant differences between positive expressed percentage of HRS markers CD15, CD30 and B cell markers CD19, CD20 in different treated L428 cells (P>0.05).3. The role of CXCL16 to HL microenvironment formation(1) There were no statistical differences between OD values of crossing Treg cells in experimental groups and control group (P<0.05), and there were also significant differences between the teated groups with adding different concentrations of sCXCL16 (P<0.05). The results confirmed the chemotaxis ability of CXCL16 to Treg cells in vitro, and this chemotaxis ability increased as the concentration increases;(2) Morphology observation under co-culture of L428 cells and Treg cells. For L428 cells, the clustering of cell group and volum of single cell were both increased, morphological change such as ellipse, short spindle-shaped and twisting were observed; while there were no significant variance of moyphology and cell nunbers of Treg cells, but the survival time of Treg cells increased;(3) Proliferation abilities of L428 cells and Treg cells under co-culture were detected by CCK-8 test. The results showed that co-culture in vitro can promote cell proliferation for these two cells;(4) Influence of recombinant IL-3 (rIL-3) to L428 cell proliferation was detected by CCK-8 test. We found that rIL-3 can promote L428 cell proliferation in vitro, and this promoting ability increased as the concentration increases.4. The molecular mechanism of CXCL16 in CHL(1) Expression levels of the key proteins such as AKT, pAKT, p70S6, p-p70S6, pIκB of PI3K/AKT/mTOR and NF-κB signal pathways in HL tissues were detected by immunohistochemistry. Phosphorylation of PI3K/AKT/mTOR and NF-κB signal pathways can be detected in CHL tissues, and phosphorylation levels of AKT and IκB were relatively weak while phosphorylation level of P70S6 was relatively strong;(2) Expression levels of the key proteins such as AKT, pAKT, P70S6, pP70S6, IκB, pIKB of PI3K/Akt/mTOR and NF-κB signal pathways in HL cell line were detected by immunocytochemistry. Phosphorylation of PI3K/AKT/mTOR and NF-κB signal pathways can be detected in HL cell line, and phosphorylation levels of P70S6 and IκB were relatively weak while phosphorylation level of Akt was relatively strong;(3) Phosphorylation levels of PI3K/AKT/mTOR and NF-κB signal pathways in L428 cells with different CXCL16 expression levels were detected by western blotting. We found that endogenously over-expressed CXCL16 and ectogenesisly adding sCXCL16 can increased the phosphorylation levels of AKT, P70S6 and IκB proteins, while ectogenesisly adding CXCL16/CXCR6 antibody can decreased the phosphorylation levels of above proteins. It was suggested that CXCL16 can activate PI3K/AKT/mTOR and NF-κB signal pathways in L428 cells;(4) The relationship between phosphorylation levels of PI3K/Akt/mTOR and NF-κB signal pathways activated by ectogenesisly adding sCXCL16 and action time was detected by western blotting. It was showed that phosphorylation level of PI3K/AKT/mTOR signal peaked at 45 minutes and for NF-κB signal it needs more than 60 minutes;(5) Influence of PI3K inhibitor LY294002 to L428 cells. Ectogenesisly adding LY294002 can suppress the proliferation and migration ability of L428 cells in vitro, and this inhibitive ability increased as the concentration increases. The promotive role of sCXCL16 to L428 cells proliferation and migration can be neutralizated when ectogenesisly combinative adding LY294002 and sCXCL16. It was suggested CXCL16 may through PI3K/AKT signal pathway to play its role to L428 cells.Conclusions1. CXCL16 and its receptor CXCR6 widely express in lymphoma; there was relatively higher expression of CXCL16 in CHL tissues and the expression of CXCL16 was related to CHL tumor size;2. CXCL16 can promote the proliferation and migration ability of L428 in vitro, decrease cell apoptosis and change cell cycle, while it has no effection on immunophenotype of L428 cells;3. CXCL16 can attract Treg cells in vitro, and the proliferation ability of L428 cells and Treg cells can be promoted by each other under co-culture;4. CXCL16 may through PI3K/AKT/mTOR signal pathway to play its role to L428 cells.New Point1. We firstly systematically researched the expression status of chemokine CXCL16 and its receptor CXCR6 in lymphoma tissues and cell lines and analysed the clinical significance of CXCL16/CXCR6 in HL tissues. Our study established the foundation for in-depth exploring of the chemokines/cytokines network of HL;2. Our study systematically analysised the role of CXCL16 and the involved intracellular signal pathway to HL through endogenously and ectogenesisly regulated the expression of CXCL16 in L428 cells, provided the clew for in-depth exploring of the intracellular molecular network of HRS cells and the potential target for biological therapy of HL;3. Our study preliminarily researched the influences of CXCL16 to the important background Treg cells and the intractions of L428 cells and Treg cells through simulating the microenvironment of HRS cells in vitro, provided the clew for explaining the formation of HL microenvironment and new thread for immunothrapy of HL. |
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