ABSTRACT
AIM: To observe the effects of NK cells stimulated with immobilized MHC class I chain-related antigen A (iMICA) on activities of dendritic cells (DCs). METHODS: Firstly fresh allogeneic NK cells, or iMICA-stimulated allogeneic NK cells, and autologous NK cells stimulated with IL-2 or IL-2 and iMICA were co-cultured with immature DCs (iDCs) at the ratio of 5:1 for 24 hours. Frequencies of HLA-DR positive or CD86 positive DCs were detected by flow cytometry. Next autologous NK cells stimulated with iMICA were co-cultured with iDCs at the ratio of 1:5 for 24 hours. Variation of HLA-DR or CD86 expression on dendritic cells was measured. Lastly IFN-γ neutralizing antibody was added into the NK-DCs co-culture system to observe HLA-DR or CD86 expression on DCs. RESULTS: When NK:iDCs ratio was 5:1, both fresh allogeneic and activated autologous NK cells killed iDCs efficiently. iMICA did not synergize this cytotoxicity. However, when NK:iDCs ratio was 1:5, NK cells activated by iMICA promoted HLA-DR and CD86 expression on DCs. IFN-γ antibody down-regulated HLA-DR and CD86 expression on DCs which were co-cultured with iMICA-activated NK cells. CONCLUSION: iMICA is redundant as fresh allogeneic or activated autologous NK cells killed iDCs. However, if numbers of NK cell are less than those of DCs, iMICA can stimulate NK cells to produce IFN-γ for DCs maturation.
Subject(s)
Dendritic Cells/immunology , Histocompatibility Antigens Class I/pharmacology , Killer Cells, Natural/immunology , Coculture Techniques , Dendritic Cells/drug effects , HLA-DR Antigens/immunology , Histocompatibility Antigens Class I/immunology , Humans , Interleukin-2/immunology , Interleukin-2/pharmacology , Killer Cells, Natural/drug effectsABSTRACT
AIM: To observe whether MHC class I chain-related antigen A (MICA) was expressed on monocytes, immature dendritic cells (iDCs), and mature dendritic cells (mDCs), and to study effect of up-regulation of MICA expression by DCs on biologic activity of NK cells. METHODS: MICA expression on monocytes, iDCs, or mDCs stimulated with LPS, TNF-α, CD40L, IL-15 or IFN-α was detected by flow cytometry. Next CD69 expression, degranuation, and IFN-γ production of NK cells stimulated with MICA-positive mDCs were analyzed. Lastly recombinant NKG2D/Fc fusion protein and anti-IL-12 monoclonal antibody was respectively added into culture systems to analyze whether these reagents affected the interaction between DCs and NK cells. RESULTS: MICA was not expressed on monocytes, and expressed on iDCs at low level. LPS, TNF-α, CD40L had no influences on MICA expression on mDCs, but IFN-α, IL-15 up-regulated MICA expression on mDCs. MICA-positive mDCs promoted CD69 expression, IFN-γ production, and killing K562 cells by NK cells. NKG2D/Fc inhibited both cytotcoxicity and IFN-γ secretion, whereas IL-12 antibody only inhibited IFN-γ secretion of NK cells. CONCLUSION: MICA expression on DCs is regulated by relevant factors in microenvironment. DCs with high level of MICA expression can up-regulate biologic activity of NK cells.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Histocompatibility Antigens Class I/biosynthesis , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Cell Line, Tumor , Flow Cytometry/methods , Histocompatibility Antigens Class I/immunology , Humans , K562 Cells , Monocytes/immunology , Monocytes/metabolism , Up-RegulationABSTRACT
OBJECTIVE: To investigate whether the major histocompatibility complex class I chain-related gene A gene (MICA) polymorphism and serum soluble MICA level were associated with the occurrence and development of colorectal cancer. METHODS: DNA samples from 117 colorectal cancer patients and 113 healthy individuals from Yangzhou in Jiangsu province were genotyped by using the polymerase chain reaction (PCR) and sequence-specific primer (SSP) method and PCR based sequencing. In addition, polymorphism at position 129 was also analyzed by PCR-SSP. Serum levels of soluble MICA were measured by a sandwich ELISA method. RESULTS: Neither the extracellular nor the transmembrane region polymorphisms of MICA gene were associated with the occurrence and the different stages of colorectal cancer. In contrast, the frequency of the methionine residue at position 129 was significantly decreased in the patient group. Soluble MICA levels in sera were increased in the late stages of colorectal cancer. CONCLUSION: Although there was no genetic susceptibility attributed to MICA gene polymorphism with regard to development of colorectal cancer, serum levels of soluble MICA may be a diagnostic marker of advanced stages.
Subject(s)
Colorectal Neoplasms/blood , Colorectal Neoplasms/genetics , Histocompatibility Antigens Class I/blood , Histocompatibility Antigens Class I/genetics , Polymorphism, Genetic/genetics , Enzyme-Linked Immunosorbent Assay , Female , Genotype , Humans , Male , Polymerase Chain ReactionABSTRACT
AIM: To study the effects of recombinant soluble MHC class I chain-related protein A (sMICA) on the cytotoxicity, secretion of IFN-gamma, proliferation and apoptosis of peripheral NK cells. METHODS: After NK cells were co-cultured with recombinant soluble MICA proteins overnight, the cytotoxicity of NK cell on target cells was detected by flow cytometry. The supernant was collected to determine the concentration of IFN-gamma by ELISA. The proliferation of NK cells to sMICA was detected by MTS/PMS. NK cells were labeled with annexin V and PI to analyze their apoptosis. RESULTS: Soluble MICA inhibited the cytotoxicity of NK cells and down-regulated the secretion of IFN-gamma, but it showed no effects on the proliferation and apoptosis of freshly isolated peripheral NK cells. CONCLUSION: The soluble MICA shedding from tumor cells could be a pathway of cancer immune evasion by down-regulating the biologic activities of NK cells.
