The Mechanism Of Dex And Tp Inhibite The Expression Of Il-4, Il-5 And Il-13 In Human Activated Peripheral Blood Mononuclear Cells

The mechanism of Dex and TP inhibite the expression of IL-4, IL-5 and IL-13 in human activated peripheral blood mononuclear cellsAsthma is a complex inflammatory disease of the lung characterized by airway narrowing, hyperresponsiveness (AHR) and inflammation. The inflammatory process, which is the basis of AHR and airway narrowing, is believed to be a result of inappropriate immune responses to common aeroallergens in genetically susceptible individuals. It has been hypothesized that lymphocytes that produce a Th2 pattern of cytokines (IL-4,IL-5,IL-13) play a pivotal role in the pathogenesis of asthma.Studies have shown that the HAT activity of the peripheral blood mononuclear cells (PBMCs) in asthmatic patients was significantly higher than that in healthy subjects, while the HDAC activity was decreased respectively. HDAC inhibitor TSA inhibited HDAC activity, and made PBMCs alter towards Th2 phenotype, in which the Th2 cytokines, IL-4,IL-5 and IL-13 were increased significantly. The study indicates that, HDAC / HAT activity may be related to the expression of the Th2 cytokines, such as IL-4,IL-5 and IL-13. However, the mechanisms underlying have yet to be clear.Costimulation of lymphocytes with anti-CD3 plus anti-CD28 antibodies can mimic the antigen activation of lymphocytes in vivo. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies to investigate the role of HDAC / HAT and IL-17A in allergic diseases,such as asthma.PART I: The dynamic changes of HDAC / HAT activity in human activated peripheral blood mononuclear cellsObjective: To investigate the effect of anti-CD3 plus anti-CD28 antibody co-stimulation on the activity of HDAC / HAT in PBMCs. Methods: PBMCs were stimulated with anti-CD3 monoclonal antibody (10μg/ml) plus anti-CD28 monoclonal antibody (5μg/ml). The HDAC / HAT activity (0min,1h,4h,24h) was measured with fluorometry. The effects of Dex and TP on HDAC / HAT activity were also studied.Results: The HDAC activity in the nucleus of PBMCs gradually decreased, and the HAT activity gradually increased, both in time-dependent manners due to anti-CD3 plus anti-CD28 co-stimulation. HAT activity was inhibited and HDAC activity was enhanced by various concentrations of Dex (10-8 M, 10-10M) and TP (100nM, 500nM) both in concentration-dependent manners.Conclusions: The increased HAT activity and decreased HDAC activity by PBMCs might play an important role in the mechanism of asthma. Dex and the TP may reduce the airway inflammation through inhibition of the HAT activity or increase of the HDAC activity.PART II: The effect of Dex and TP on the release of Interleukin-4, 5 and 13 in human peripheral blood mononuclear cellsObjective: To investigate the effect of anti-CD3 plus anti-CD28 antibody co-stimulation on the release of Interleukin-4,5 and 13 in PBMCs. And the effect of Dex and TP on IL-4,IL-5 and IL-13 release was also studied. Methods: PBMCs were stimulated with anti-CD3 monoclonal antibody (10μg/ml) plus anti-CD28 monoclonal antibody (5μg/ml). ELISA was employed to measure the concentration of IL-4,IL-5 and IL-13 in the supernatant of the cell culture (0min,24h,48h,72h); IL-4,IL-5 and IL-13 mRNA levels in PBMC in 0min,6h,12h,24h were measured by Real-time PCR.Results: IL-4,IL-5 and IL-13 were released in a time-dependant manner in PBMCs due to anti-CD3 plus anti-CD28 co-stimulation, however the release could be inhibited by Dex(10-8M)and TP (100nM,500nM). Conclusions: IL-4,IL-5 and IL-13 produced by PBMCs might play an important role in the mechanism of asthma. Dex and TP might inhibit the Th2 type allergic inflammation in asthma through reducing the release of the IL-4,IL-5 and IL-13. Dex and TP possibly inhibites the expression of IL-4, IL-5, IL-13 and other inflammatory gene through inhibiting the HAT activity and increasing HDAC activity.PART III: The Effect of Triptolide on the release of IL-17A and IFN-γin human peripheral blood mononuclear Cells Objective: To investigate the effect of anti-CD3 plus anti-CD28 antibody co-stimulation on the release of IL-17A and IFN-γin PBMCs. And the effects of Triptolide on IL-17 A and IFN-γrelease were also studied. Methods: PBMCs were stimulated with anti-CD3 monoclonal antibody (10μg/ml) plus anti-CD28 monoclonal antibody (5μg/ml). ELISA was employed to measure the concentration of IL-17A and IFN-γin the supernatant of the cell culture. Results: IL-17A and IFN-γwere released in time-dependant manners in PBMCs due to anti-CD3 plus anti-CD28 co-stimulation, however the release could be inhibited by Triptolide at the concentration of either 100nM or 500nM. Conclusions: IL-17A produced by PBMCs might play an important role in the mechanism of asthma. Triptolide might inhibit the Th2 type allergic inflammation in asthma through reducing the release of the IL-17A .Comprehensive results of the above three parts, we can conclude:①HDAC / HAT activity may be related to the expression of the Th2 cytokines, such as IL-4,IL-5 and IL-13;②Dex and TP possibly inhibites the expression of IL-4, IL-5, IL-13 and other inflammatory gene through inhibiting the HAT activity and increasing HDAC activity ;③TP might inhibit the Th2 type allergic inflammation in asthma through reducing the release of the IL-17A.