| BackgroudLymphoma is a malignant tumor dereived from Lympho-hematopoietic tissue, including Hodgkin’s lymphoma and non-Hodgkin’s lymphoma.Classical Hodgkin’s lymphoma (cHL) is characterized by only a few malignant cells and an abundance of inflammatory cells. Hodgkin and Reed-Sternberg (H/RS) cells are surrounded by T and B cells admixed with plasma cells, macrophages, eosinophils and mast cells,and contribute to the composition of the special microenvironment in cHL. Not only H/RS cell but also the microenvironment should be focused on during the research of Hodgkin lymphoma.It is reported that probably a variety of cytokines support the proliferation and apoptosis suppression of H/RS cells. Most of the cells are T cells in the background of H/RS cells. Surprising, the tumor cells are not killed by the T cells but survive. T lymphocytes may play an essential role in maintaining the survival of H/RS cells. The interaction between tumor cells and immune cells including T cells may provide a suitable living environment for H/RS cells by various cytokines/chemokines.To explore the role of cytokines/chemokines on cHL, we used online software GeneSifter and BRB-Array Tools to analyze the differences between cHL cell line (L428), Burkitt’s lymphoma cell line (Raji), H/RS cells and Diffuse large B-cell lymphoma (DLBCL) cell gene expression data in GEO public database, tried to find the closely related cytokine/chemokines with the H/RS cells,results show that high expression of RANTES/CCL5, CCL17, CCL22, SISd, IL-6 and CXCL16,among which CXCL16 cause our attraction.The chemokine CXCL16 is produced by defensive immune cells and differs from the other known soluble chemokines by its structure:exists in both transmembrane (TM-CXCL16) and soluble forms (sCXCL16). CXCL16, normally expressed in Thl cells, NK cells and activated CD8+T cells, has been associated with the migration of effector T cells, interactions of APC and CD8+T cell, and response of cellular immune and inflammation as well as growth of thymocytes. Study founded that CXCL16 plays an important role in migration of leukocyte of human inflammatory diseases including atherosclerosis, rheumatoid arthritis and HIV infection diseases.As the similarities between tumor cell migration and leukocyte migration, chemokines play a decisive role in tumor organs selective metastasis and cancer progression, CXCL16 has recently been focused on. More foreign research on CXCL16 expression in prostate cancer, breast cancer, colon cancer, and pancreatic adenocarcinoma cells and human glioma cells, CXCL16 may be present in tumor cells and keeps a closely relations with tumor invasion and metastasis, as well as conditioned to tumor cell growth, adhesion and directional migration. Also be present in the infiltrating immune cells involved in the interaction between tumor cells and the immune system, however, the relationship with the development of tumors has not been fully elucidated.In 2009, Darash-Yahana detected that CXCL16 expression in 25% of cases in 28 cases of lymphoma. As lymphoma could be divided into different types and subtypes with different histological characteristics, it is necessary to investigate the expression of CXCL16 on lymphoma of different origins. Hitoshi founded that both CXCL16 and its receptor CXCR6 were expressed on HL cells, however, it was not yet known that whether there are different expressions between H/RS cell and cell lines of other different origins.Foreign studies and ours have shown that the abnormal increased NF-κB activity and lack of human CD99 gene are related to the occurrence and development of H/RS cell. The relationship between CD99 and CXCL16 expression remained to be illustrated, besides, whether NF-κappaB signaling pathway is involved should be discussed in order to prolong our previous research.Previous reports showed that CD4+T cells is the main componant of cHL microenvironment. Whether the survival of H/RS cells is affected by the effect of CXCL16 or through the interaction between the tumor cell and CD4+T cell is also worth being explored. Thus the objectives of this study are as follows:Objectives(1) Defining the expression of CXCL16 and its receptor CXCR6 in cultured lymphoma cell lines, analysing the characteristic of CXCL16 expression in cell lines of different orgins.(2) Observing the relationship between CXCL16 and CD99 expression and whether the NF-κappaB signaling pathway is involved.(3) Detecting the proliferation of L428 cell and chemotactic ability of peripheral blood CD4+T cell on the recombinant CXCL16, analysing the possible role of CXCL16 in the H/RS microenvironment in vitro.Methods:1. The expression of CXCL16 and its receptor CXCR6 in lymphoma cell lines of different orgins.Expression of CXCL16/CXCR6 in seven B-cell origin and two T-cell lymphoma cell lines was detected by real time Quantitative PCR(Q-PCR), western blotting, confocal microscopy and Enzyme linked immunosorbent assay (ELISA),These cell lines were cHL cell line(L428), diffuse large B cell lymphoma cell lines (OCI-Ly1, OCI- Ly8,and OCI- Ly10),Burkitt’s cell line(Raji), multiple myeloma cell lines (RPMI-8226 and KM3), anaplastic large cell lymphoma cell line (Karpass299) and acute T cell leukemia cell line(Jurkat)2. The relationship between CXCL16 and CD99 and the role of NF-κB signaling.Lymphoma cell morphology was observed in previously established subcell lines (L428-CD99+ and A20-mCD99L2-), RNA extraction and mRNA expression of CXCL16 and CD99 by real time RT-PCR was analysised and CXCL16, CD99 protein by Western blotting was detected. The secreted CXCL16 protein by enzyme linked immunosorbent(ELISA), and the expression of CXCL16, CD99 and p-IκBα protein by Western blotting was detected. The relation of CXCL16 and CD99 expression was defined as the same time. The expression of CXCL16 and CD99 by regulating the NF-κappa B signaling pathway through the NF-κappaB activator and inhibitor used was observed.3. The role of CXCL16 on the microenvironment of classical Hodgkin lymphoma in vitro.CD4+T lymphocytes were isolated from human peripheral blood mononuclear cells (PBMC) by Human CD4 immunomagnetic and co-cultured with L428 cells to simulation the microenvironment in vitro. Co-cultured L428 cell proliferative capacity was detected by CCK8. To detect their chemotactic effects on CD4+T cells, L428 supernatants,human CXCL16 cell factor and its antibody in different combinations as the conditioned medium (conditioned media, CM)were applied to analyze the role of chemotactic factor sCXCL16 simulation of the H/RS cell microenvironment in vitro.4. Statistical analysisStatistical software SPSS 13.0 was used to analysis the experimental results. Measurement data were expressed as mean ± standard deviation (x ± s). Results from Real-time PCR, flow cytometry and CCK8 proliferation assay were analyzed by using single-factor analysis of variance (One-Way ANOVA). LSD method was used to compare differences between groups. The level of α=0.05, P<0.05, a statistically significant difference.Results:I. The expression of CXCL16 in lymphoma cell lines.1. Results from Quantitative PCR showed that compared to that in L428 cell which is valued as 1, the expression of CXCL16 mRNA in OCI-Lyl, OCI-Ly8, OCI-Ly10, Karpass299 and Raji cells are 0.454± 0.024,0.430±0.046,0.443±0.046, 0.233±0.054 and 0.219±0.047 folds, respectively. The mRNA expression of CXCL16 in L428 cell is higher than those of OCI-Ly1, OCI-Ly8, OCI-Ly10, Karpass299 or Raji cell, with statistical significance (P<0.05), respectively.The mRNA expression of CXCL16 is 4.115±0.234,4.378±0.339 and 5.687±0.194 folds in RPMI-8226,KM3 and Jurkat cells compared to that of L428 cell. The mRNA expression of CXCL16 in L428 cell is lower than those of RPMI-8226,KM3 or Jurkat cell, with statistical significance (P<0.05) respectively.Results from Quantitative PCR showed that compared to that in L428 cell which is valued as 1, the expression of CXCR6 mRNA in OCI-Ly1,OCI-Ly8 and OCI-Ly10, Karpass299, Raji and Jurkat cells are 6.946 ± 0.781,7.762 ± 0.357,7.330±0.794, 15.702 ±1.495,5.068 ±0.785 and 9.548 ±1.284 folds, respectively. The mRNA expression of CXCR6 in L428 cell is lower than those of OCI-Lyl or OCI-Ly8 or OCI-Ly10 or Karpass299 or Raji or Jurkat cell, with statistical significance (P<0.05), respectively. The expression of CXCR6 mRNA in RPMI-8226, KM3 cells are 1.260 ± 0.301 and 1.765 ± 0.096 folds. The mRNA expression of CXCR6 in L428 cell is higher than those of RPMI-8226 or KM3 cell, with statistical significance (P<0.05), respectively.2. Results from Western blotting revealed the CXCL16 protein in multiple plasma cell myeloma cell lines RPMI-8226, KM3 and the acute leukemia lymphoma cells Jurkat were highly expressed, relatively high expression in L428 cell,and low expression in DLBCL cell lines OCI-Lyl,OCI-Ly8 and OCI-Ly10, anaplastic large cell lymphoma Karpass299 and Burkitt’s lymphoma cell line Raji. CXCR6 protein expression in DLBCL cell lines OCI-Lyl,OCI-Ly8 and OCI-Ly10, Karpass299, Raji and Jurkat cells is higher,or lower expression in L428, RPMI-8226 and KM3 cells.3. Results from immuno-fluorescence detection showed that TM-CXCL16 protein were higher expressed in RPMI-8226, KM3 and Jurkat cells while lower expressed in DLBCL cell lines OCI-Ly1,OCI-Ly8 and OCI-LylO, Karpass299 and Raji than in L428 cells,which is consistent with results from Western blotting analysis.4. Results from ELISA revealed that the amounts of sCXCL16 secretion in OCI-Lyl,OCI-Ly8,OCI-LylO,Karpass299,Jurkat,Raji cells were higher than in L428 cell, while lower in RPMI-8226,KM3 cells than in L428 cells, with significant differences between them (P<0.05).Ⅱ. The relationship of CXCL16, CD99 and NF-κB signaling pathway1. During continuous passage, A20-mCD99L2 cells were found to become into large cells with aboundant cytoplasm and there are more dual-neuclei or multi-neuclei cell appeared, while large cells in the L428-CD99+ cells significantly decreased and cell morphology tend to be more consistency.2. The mCD99 mRNA of A20-mCD99L2-cells was 0.331±0.093 times than that of A20 cells, with statistical significance (P<0.05). The mCXCL16 mRNA of A20-mCD99L2-cells was 2.440±0.156 folds than that of A20 cells, with statistical significance (P<0.05). The hCD99 mRNA of L428-CD99+ cells was 3.807±0.763 times than that of L428 cells, with statistical significance (P<0.05). The hu-CXCL16 mRNA of L428-CD99+cells was 0.034±0.011 folds than that of L428 cells, with statistical significance (P<0.05).3. The CXCL16 protein of A20-mCD99L2- cells is higher than that of A20 and A20-con cells while that of L428-CD99+ cells is below L428 and L428-con cells by western blotting.4. mCD99L2- protein in A20-mCD99L2- cells was weaker than in A20 cells while p-IκBα protein stronger than in A20 cells. Applying NF-κB signaling pathway inhibitors BAY on A20-mCD99L2- cells had no effect on mCD99 protein, but significantly decreased protein expression of p-IκBα and CXCL16, Applying NF-κB signaling pathway activator LPS on A20 cells had no effect on mCD99 protein, but increased protein expression of p-IκBα and CXCL16.hCD99 protein in L428 cells was weaker than in L428-CD99+ cells. Applying BAY on L428 cells had no effect on hCD99 protein, but significantly decreased the p-IκBα and CXCL16 expression. hCD99 protein was not affected in L428-CD99+ cells treated with LPS but the p-IκBα protein and CXCL16 were enhanced.5. ELISA detection revealed that in A20-mCD99L2- cells treated with NF-κB signaling pathway inhibitor BAY,the secretion of CXCL16 was 429.921±27.495, significantly reduced, with statistical significance (P<0.05). In A20 cells treated with NF-κB signaling pathway activator LPS, CXCL16 secretion was significantly increased (716.202±89.89), with statistical significance (P<0.05). In L428 cells treated with inhibitors BAY, the secretion of CXCL16 was 241.528±49.169, significantly reduced than before, with statistical significance (P<0.05).In L428-CD99+ cells treated LPS, secreted CXCL16 was 624.809±42.643, significantly increased, with statistical significance (P<0.05).III. Primary role of CXCL16 on cHL in vitro.1. L428 cells were co-cultured with CD4+T cells and cell proliferation ability was analyzed by CCK8 assay. There were significant difference among the different cell groups (F=828.887,P<0.05), LSD multiple comparison showed that the proliferation activity of co-culturedL428 cell was significantly higher than that of control group (P<0.05)2. Recombinant protein CXCL16 was added in L428 cells and cell proliferation ability was analyzed by CCK8 assay. The results showed that there were no significant difference between different cell groups (F=3.782,P>0.05). LSD multiple comparison showed that the proliferation activity of experimental group has no significantly higher than that of control group (P>0.05);3. Chemotaxis ability was deteced by Chemotaxis assay. L428 cells in serum-free culture supernatant was as a control, the results suggested that when concentration of recombinant CXCL16 protein was lOng/ml,20ng/ml,30ng/ml, the number of CD4+T cells attracted was 93980.00 ±62516.656,154229.3 ± 55021,418 232090.3 ± 61388.819 times compared with the control group. In the 30ng/ml group, with significant difference in 10ng/ml group (P<0.05).Conclusions1. CXCL16 and its receptor CXCR6 were widely expressed in lymphoma cell lines with different origins. CXCL16, including TM-CXCL16 and sCXCL16, was relatively high expressed in classic Hodgkin’s lymphoma L428 cells while CXCR6 lower expressed.2. Expression and secretion of CXCL16 were negatively correlated with CD99/ mCD99L2 and affected by the regulation of the NF-KappaB signaling in cHL cell lines L428 and murine A20 cells.3. Hunman recombinant CXCL16 does not affect the proliferation of H/RS cells in vitro but promoted chemotaxis of CD4+T cell.New Point1. Using a variety of methods in vitro, the differently expressed CXCL16/CXCR6 in tumor cell lines of different origins were detected systematiclly for the first time, providing the basis for further studing and potential target for biological treatment of lymphoma.2. By analyzing the relationship between CXCL16 and CD99 study, our previous studies on CD99 was extended in this study. By regulating NF-κappa B signaling, the relationship between CXCL16, CD99 and NF-KappaB were also explored, which may contributing the research of chemokine network and pathway studies.3. By isolating the CD4+T lymphocyte and co-cultureing with L428 cells, which providing the method simulating the microenvironment of H/RS cell in vitro,the interaction between tumor cells and the microenvironment and the significance of CXCL16 were primarily studied. |
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