Studies on EBV-specific Antibody Responses and EBV-regulated Cytokine Expression in Nasopharyngeal Carcinoma Patients

• Main Authors: Yu-Tzu Huang, 黃毓慈
• Other Authors: Chi-Hwa Tsai
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/51628208357439036931

博士 === 國立臺灣大學 === 微生物學研究所 === 89 === Nasopharyngeal carcinoma (NPC) is an epithelial cancer that is causally associated with Epstein-Barr virus (EBV) infection. The roles of EBV-related immune responses in nasopharyngeal carcinoma (NPC) individuals and in EBV-infected healthy donors were interested for studying. Experimental designs for the tests of antibody responses were using baculovirus-expression system as target cells for immunofluorescence assay and cellular immunity examination was performed by 3H-thymidine incorporation cell proliferation assay. Seventy NPC patients and 32 control individuals were examined in this study. According to data of immunofluorescence analysis, the antibody titers to EBV-encoded BMRF-1 gene products (52/50 kD diffused early antigen, EA-D), DNase, and thymidine kinase were higher in NPC patients than those in control individuals, especially for IgA type. The elevated IgA antibodies for all three EBV lytic antigens were detectable in NPC individuals but not in the controls mean. The most interesting finding was that antibody titers to these EBV lytic proteins were significantly associated with tumor progression of cervical lymph node metastases. Results of cell proliferation assay revealed that peripheral blood cells of NPC patients could not be prominently stimulated by either purified EA-D or DNase protein, except in few NPC individuals who usually had very high titers antibodies against EBV encoded-EA-D and -DNase. NPC tumor biopsies are characterized histopathologically by an abundant infiltration of non-malignant lymphocytes, such as T cells and B cells. Based on the cytokines involving with the antibody production of B cells, T cells activation and tumor growth, we analyzed the expression of various cytokines in NPC tissues in order to investigate the interaction of the infiltrating lymphocytes and EBV-harbored tumor cells in the part II study. Analysis using the reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the expression of a panel of cytokines in the NPC biopsies: interleukin-1a (IL-1a), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, interferon-g, tumor necrosis factor-a, transforming growth factor-b and IL-1 receptor types I and II. Elevated expression of IL-1a and IL-1b was observed in primary tumors and NPC metastases compared to control tissues. Interestingly, this increased expression correlated with the EBV-encoded viral IL-10 transcript. To determine which cells were responsible for producing IL-1, the cellular constituents of NPC biopsies were determined by immunoflow cytometric analysis. Based on the data from these analyses, the three major specific cell populations: epithelial, CD4+ T cells and CD8+ T cells from 5 NPC tumors were selected using specific, antibody-coated paramagnetic beads. RT-PCR of RNA from these fractionated cells showed that transcripts of IL-1a and IL-1b were present, not only in the malignant epithelial cells, but also in CD4+ T cells infiltrating the tumor, a finding confirmed by immunohistochemical staining. We expected that the unusual synthesis of IL-1a and IL-1b by EBV-positive epithelial cells might be responsible for presence of EBV. We expected that elevated production of IL-1 was associated with the presence of EBV in epithelial cells. Therefore, the aim of part III study is to determine which EBV products contribute to the production of IL-1. We analyzed induction of IL-1 mRNA using RT-PCR in a panel of epithelial cells that stable expressed EBV-encoded nuclear antigen 1 (EBNA1), latent membrane protein (LMP) 1 and LMP2A. The result showed that LMP1 was sufficient to induce IL-1 mRNA, whereas, expression of individual EBNA1 or LMP2A resulted in no significant effects on the production of IL-1a and IL-1b messages. Transient expression of LMP1 in epithelial cells resulted in induction of IL-1 confirmed by quantitative RT-PCR. The secreted IL-1a and IL-1b were also detected in the culture supernatants of the LMP1 transfectants by specific ELISA assays. In addition, raising culture temperature to 42oC or treating with tumor necrosis factor-a enhances LMP1-mediated IL-1 production. These results suggested that LMP1 combining with environmental factors might regulate IL-1 production in epithelial cells. Furthermore, matrix metalloproteinase-2 and -9 were not responsible for IL-1 stimulation, but IL-1a and IL-1b have the ability to upregulate the growth of epithelial cells. This study provides detail informations about EBV-regulated cytokine expression and EBV-specific antibody responses and cellular immune responses in NPC. It hypothesizes that the cytokine secreted via infiltrated lymphocytes or EBV pathway may contribute to tumor progression and growth during NPC development.