The Opposite Effect Of IFN-α And IFN-γ On Expression Of MHC Class I Chain-Related A And The Research For Its Mechanism

Objective NK cells constitute an important component of the innate immune system, providing surveillance against certain viruses, intracellular bacteria and transformed cells. NK cells exert cell-mediated cytotoxicity and act to regulate innate and acquired immune responses through the release of various cytokines ( such as IFN-γ, GM-CSF and TNF-β) and chemokines. Therefore, they stand as a bridge between innate and adaptive immune responses. Unlike T cells, NK cells killing of virus-infected or malignant transformed cells do not need pre-sensitization and are independent of a MHC restricted manner, thus take a vital position in both innate and acquired immunity. Therefore, NK cells are now the hot items in immunological researches and are considered as promising reagents in adoptive transferring treatment for cancers especially haematopoietic disorders.NK cells survey the MHC class I expression by inhibitory receptorys on target cells and eliminate cells with aberrant MHC class I expression. In the absence of MHC class I expression, NK cells are release from the negative influence of MHC class I expression and kill the target. However, the existence of target cells with normal MHC class I expression that are sensitive to NK cells suggests that there may be other mechanisms to regulate NK cell activity. The activating receptors also play major roles and are believed to be necessary for the initial activating of NK cells. The well-characterized example to date is the NKG2D receptor and its ligands. Thus, NK cell effector functions are regulated by integrated signals across the array of stimulatory and inhibitory receptors engaged upon interaction with target cell surface ligands. NKG2D is an activatory immunoreceptor whose expression was first recognized on NK cells, but was subsequently found on CD8+αβT,γδT and Macrophages, making it one of the most widely distributed NK receptors currently described. In human, the NKG2D receptor binds to the stress-inducible polymorphic nonclassical MHC molecules MICA (MHC class I chain-related protein A), MICB and the UL16-binding proteins-l, 2, 3 (ULBP-l, 2, 3), which are up-regulated strongly in many types of tumor cells. Recent studies reveal that the expression of MIC and ULBP on human tumor cells is sufficient to overcome the inhibitory effects of MHC class I expression on NK cell killing. These observations indicate that NKG2D provides first line surveillance against stressed or abnormal cells that have been induced to express one of its ligands.Interferon is an important regulator of the immune response to tumors. Type I interferons (IFN-αand IFN-β) exert many biological functions including antiviral activity, anti-proliferation and anticancer effects. They do not directly kill tumor or infected cells, but exert immunoregulatory functions on immune cells to eliminate transformed or virus infected cells. Type II interferon, immune interferon, IFN-γis a multi-functional cytokine produced by Th1 and NK cells. It increases the expression of Fc receptors for IgG on macrophages and PMNs as well as increasing MHC class I and II expression on a wide variety of cells. This enhances the phagocytic function of these cells as well as increasing the antigen presenting capabilities of professional antigen presenting cells. More importantly, IFN-γfunctions to regulate Th1 responses that are critical to CTL responses and IgG antibody production. Also, it is a critical component of the endogenous and many cytokine-induced antitumor immune responses. Recent studies reveal that the 5'end flanking regions of MICA and MICB contain putative heat shock elements (HSE), which are prototypic transcription inducer sites in heat shock protein 70 (HSP70) genes that bind activated trimeric heat shock factor 1 (HSF1). Moreover, IFN-αand IFN-γexert opposite effect on the expression of HSP70. IFN-αcan up-regulate the expression of MICA in dendritic cells. However, the direct effect of IFN-αand IFN-γon the expression of MICA in human tumor cells is still unclear, and the mechanism of MICA gene expression need more research.In this study, on one hand, we observed the effects of IFN-γand IFN-αon the expression of MICA in different tumor cells ( HeLa, Caski, K562, Raji ) as well as the cytotoxicity of peripheral blood NK cell to them. The aim is to investigate whether IFN-γand IFN-αcan oppositely regulate the expression of MICA , then exert opposite effects on the susceptibility of the carcinoma cells to NK cytolysis. On the other hand, we constructed luciferase reporter vector pGL3/MICA-Promoter by gene engineering technology and observed the effect of IFN-γand IFN-αon the transcriptive activity of MICA promoter region. Then, we observed the effect of IFN-γon metalloproteinase(MMP) and tissue inhibitor of metalloproteinase(TIMP) expression, and observed the release of soluble MICA (sMICA) in supernatants. The aim is to investigate how IFN-γand IFN-αregulate MICA gene express on transcription and post-translation level.Methods(1) The cytotoxicity of human peripheral blood NK cells was detected by MTT method. (2) The expression of MICA was measured by RT-PCR, or Immunohistochemical mechod (SP technique). (3) Construction of MICA pormoter clone vector. MICA promoter DNA was obtained from human cervical carcinoma cell line Hela by PCR method and was sequenced by cloned to plasmid pMD18-T vector. (4) Construction of MICA pormoter luciferase reporter vector. The recombinant plasmid pMD18-T/MICA-Promoter was digested with Xhol and HindIII, then MICA pormoter fragment was isolated and inserted into the corresponding restriction site on luciferase reporter vector pGL3-Basic. (5) The lipofectamine was used to co-transfect luciferase reporter plasmid (pGL3 and pRL-TK) into the HeLa and Caski cells. The transcriptive activity of MICA promoter region was measured by using the Synergy HT Multi-Detection Microplate Reader to run the dual-luciferase reporter assay system. (6) The expression of MMP and TIMP was measured by RT-PCR method, and the release of soluble MICA (sMICA) in supernatants was measured by ELISA method.Results1. The effect of IFN-αand IFN-γon expression of MICA. RT-PCR results showed, compared to Caski cells and Raji cells which experessed week and negtive level of MICA respectively, HeLa cells and K562 cells experessed relatively higher level of MICA. IFN-αenhanced the mRNA expression of MICA in all the four tumor cells, while IFN-γinhibited it in the MICA+tumor cells in dose and time dependent manner. These results were confirmed by Immunohistochemical assay that IFN-γand IFN-αexert opposite effect on MICA expression.2. The effect of IFN-γand IFN-αon the susceptibility of the tumor cells to NK cytolysisIFN-αcan promote the susceptibility of all the four tumor cells to NK lysis, while IFN-γinhibited the susceptibility of the MICA+ tumor cells to NK lysis. Antibody blocking assay showed that the surface expression of MICA on tumor cell are responsible for the effect of IFN-γand IFN-αon the susceptibility of the tumor cells to NK cytolysis.3. Cloning of MICA promoter DNA.Total DNA was isolated from fresh culturing HeLa cells. PCR was amplified with specific primers for MICA promoter. After PCR product was detected by ethidium bromide-stained agarose gel electrophoresis, it was then lingased with pMD18-T vector, cloned and sequenced.The sequence of cloned MICA promoter DNA was identical with those in Gene Bank.4. Construction of the vector pGL3 / MICA-promoterThe MICA promoter fragment was digested by XhoI and HindIII from pMD18-T/MICA-promoter. Purified fragment was cloned into corresponding sites of the pGL3/Basic. Then, the recombinant vector was transformed into e.coli JM109. The postive clone which were screened by PCR and identified by restriction endonucleases was the recombinant reporter vector of pGL3 / MICA-promoter.5. The effect of IFN-γand IFN-αon the activity of MICA promoterThe dual-luciferase reporter vector pGL3 and pRL-TK was co-transfected into HeLa and Caski cell lines by lipofectamine regeant. After treated with IFN-γand IFN-α, the firefly luciferase activity and renilla luciferase activity was measured by using the Synergy HT Multi-Detection Microplate Reader to run the dual-luciferase reporter assay system. The rusults show that IFN-α(1000 u/ml, 48h) can improve the promoter transcripctive activity, while there were no influence on it by IFN-γ.6. The effect of IFN-γand IFN-αon the release of soluble MICAIFN-γenhanced the mRNA expression of membrane type matrix metallo- proteinase-2(MT2-MMP) and metalloproteinase9(MMP9), and down-regulated the mRNA expression of the tissue inhibitor of metalloproteinase 1 (TIMP1), while there were no influence on it by IFN-α. Moreover, IFN-γtreatment (1000 u/ml, 48h) can increase the level of sMICA in cultural supernatants of K562 and HeLa cells. Conclusions1. IFN-αcan promote the susceptibility of the tumor cells to NK lysis by increasing the surface expression of MICA2. IFN-γhas an opposite effect on the surface expression of MICA and has a negative effect on the susceptibility of the MICA +tumor cells3. IFN-αup-regulated MICA gene expression though increasing the promoter transcripctive activity.4. IFN-γdown-regulated the surface expression of MICA, and up-regulated the resease of soluble MICA (sMICA) in supernatants by promoting the cleavage by MMP.We postulated theories for the first time that"IFN-αand IFN-γexert opposite effect on the expression of human NKG2D ligands-MICA". This research provided foundation for exploring factors that control the expression of NKG2D ligands and then regulate the balance between activating and inhibitory signals. Modulation the balance between activating and inhibitory signals through NKG2D ligands may open new approaches to NK cell-based biotherapy for cancer and infecious diseases.