| Natural killer(NK) cells and CD8+ T cells composed the main effector cells of the native and adaptive immune response.When activated,they both exhibit the function as cytolysis and cytokines releasing;Among cytokines secreted by NK cells or T cells,IFN-γattracted more attention not only for it's direct anti-virus activities but also the immunoregulatory functions.Activation-related immunoreceptor NKG2D is found expressed on NK cells,CD8+ T cells,γδTCR+ T cells,as well as the macrophages.Upon ligation with it's ligands, NKG2D initiate full activation signals for NK cells and macrophages,but co-stimulating signals for CD8+ T cells in the presence of TCR stimulating.Among the NKG2D ligands, stress-inducible MHC class I chain related(MIC) molecules MICA and MICB are the first identified and also the best characterized.Cells expressing MIC molecules on their surface are susceptible to NK and T cell immunity.MICs has been observed up-regulated or induced on the surface of different cells in response to heat shock,oxidative stress, infection,and transformation.In normal humans,MICs is transcribed in keratinocytes, endothelial cells,fibroblasts,monocytes,epithelial cell lines and in gastrointestinal epithelial tissues.However it is not usually transcribed in T cells,B cells,and also NK cells. MICs protein has been found present at the cell surface of endothelial cells,fibroblasts,and epithelial cells in low level,or exist in the cytoplasm of monocytes,kerotinocytes and activated T cells.Monocytes circulating in the blood stream are normally immunologicaly quiescent. However,under certain conditions they contribute to immune responses through differentiating into dendritic cells or secreting large amouts of cytokines.Recent reports even described a novel reciprocal activation between monocytes and lymphocytes;The cytokine IFN-γderived from NK cells or T cells,as well as the cell-cell contact seem to be the key factors involved in those cells cross-talk.In the regarding of the important role of NKG2D receptor in NK/T cell triggering and the role of NKG2D receptor/ligand system in cross-linking lymphocytes and antigen presenting cells(APCs),we presumably think NKG2D might mediate the interaction between monocytes and NK/T cells.Although the immune response initiated by the T-cell response to viral antigens is fundamental for hepatitis B virus(HBV) clearance,NK cells are also essential for the initial control of HBV replication and the later induction of HBV-specific CTLs.The compromised function and decreased number of NK cells in chronic HBV infection has been observed,yet the underlied mechanism of NK cells functional defects still remain elusive.Additionally,IFN-γhas been proposed the most pivotal factors for host successive clearance of HBV,but exogenous IFN-γexhibits less efficience in the theapy of patients with chronic HBV infection.The precise reason is also not well understood.To explore the role of NKG2D receptor/ligand system in the reciprocal interaction between NK/T cells and monocytes,and clarify the underlied mechanism causing functional impairement of NK cells and unresponsiveness to exogenous IFN-γin patients with chronic HBV infection,follows work were performed in this research:1.The morphologic and phenotypic changes of monocytes in the response of IFN-γ,TNF-α,and IFN-αstimulation were observed by microscope and flow cytometric analysis respectively; 2.The surface expression of MICs on PBMCs,isolated primary monocytes,as well as the monocytic cell line received various stimuli were examined by flow cytometry;3.RT-PCR and western blot were further used to determine the expression of MICA and MICB mRNA and protein in monocytes with or without IFN-γstimulation;4.IFN-γprestimulated monocytes were cocultured with allogenic NK cells,then NK cell activating marker of the surface CD69 and the intracellular IFN-γexpression were examined by flow cytometry,and the cytolytic capacity of NK cells against K562 cells was studied by 51Cr-releasing assay;5. freshly isolated monocytes were subjected to treatment with the supernatant collected from the activated NK cells,monocytes surface expression of MICs and mIL-15 were analyzed by flow cytometry and then subjected to the coculture with CD8+CD28-T cells.CD8+CD28-T cells activating marker CD25 and CD62L were then detected by flow cytometry;6.The NKG2D expression level on NK/T cells were also assayed in the presence of anti-IL-15 antibodies or anti-MICs antibodies blocking after coculture with monocytes.7.The capacity of promoting normal NK cells activating by IFN-γ-prestimulated monocytes collected from patients with chronic HBV infection were assessed;and the property of MIC and mIL-15 induction in the response of IFN-γstimulation were also surveyed in 15 healthy donors and 13 patients with chronic HBV infection.We demonstrated here that:1.After IFN-γstimulation,monocytes displayed the shape of fusiform or polygon and were more likely to adhere and cluster to form the distinct structure as "cell islet";IFN-αweakly promoted monocytes cluster and induced monocyte form lots of projections on the surface;TNF-αtreated monoctyes showed no visible morphologic changes;2.Both IFN-γand IFN-αinduced or up-regulated the surface expression of CD80,CD83,CD86,and HLA-DR on monocytes,concomitantly down-regulated the CD14 expression;IFN-γeven induced the CD1a expression on monocytes after 5 days stimulation;Monocytes cultured in vitro for 5 days in the presence of IFN-γchange their phenotype from CD14+CD1a-CD83- to CD14+CD1a+CD83+;TNF-αseemed can no impact monocyte phenotype;3.Freshly isolated monocytes displayed no or low level expression of MICs on the surface as assessed by flow cytometry;RT-PCR and western blotting revealed that both transcripts and protein of MICA and MICB exist in the lysates of freshly isolated monocytes.After IFN-γstimulation,MICs is exclusively induced or up-regulated on the surface of monocytes within PBMCs;Surprisingly,both MICA and MICB are not responsible for this up-regulation because neither anti-MICA nor anti-MICB mAB exhibited the enhanced positive staining,and this was confirmed by western blotting analyzing MICA and MICB protein expression in monocytes with or without IFN-γstimulation.4.IFN-γtreated monocytes enhanced IFN-γproduction,CD69 expression,and K562 cytolytic ability of NK cells.MICs-NKG2D interaction between NK and monocyte seem to be responsible for the NK activation,because mAB-mediated masking of MICs as well as inhibition of cell-to-cell contact using transwell insert significantly abolished NK cell activation.Meanwhile,IFN-γsecreted by activated NK cells also conferred monocytes the capacity of activating CD8+CD28- T cells in the presence of anti-CD3 stimulation;5. The enhanced expression of MICs was associated with the up-regulation of membrane-bound IL-15(mIL-15) expression on monocytes,which elicit the role of preventing NK cells or CD8+CD28- T cells from MICs-induced NKG2D down-modulation in the contact with monocytes;6.Finally,monocytes recovered from chronic HBV-infected patients showed defects in MICs and mIL-15 surface induction and impaired ability to activate NK cells in response to IFN-γstimulation.Based on these findings we can draw a conclusion that lymphocytes-derived IFN-γmay exerts it's immunoregulatory role through driving monocytes differentiate to mature dendritic cells(DCs) and up-regulating a novel unknown MIC molecule and mIL-15 expression on monocytes;The up-regulated MIC and mIL-15 were then synergistically enhance NK/T cells activities.The defects of MIC and mIL-15 induction on monocyte might contribute to NK cells functional compromise in chronic hepatitis B virus infection. |
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