| PART ?IMPACT OF CIGARETTE SMOKE EXTRACT AND CD40-CD40L PATHWAY ON MURINE DENDRITIC CELLS INDUCING CD4+T CELLS POLARIZING TO TH1/TH2 CELLSABSTRACTObjective:To observe the impact of cigarette smoke extract(CSE)on dendritic cell differentiation Thl/Th2 cells,and explore the role of CD40-CD40L signal pathway in effect of cigarette smoke extract on Dendritic cells inducing CD4+T cells polarizing to Thl/Th2 cells.Methods:About 50 SPF class male balb/c mice,aged 6 to 8 weeks,weight of 22g to 25g,Dendritic cells(DCs)had been generated from bone marrow-derived mononuclear cells of these mice,by which culture in RPMI1640 medium containing 10%fetal bovine serum(FBS)with granulocyte-macrophage colony-stimulating factor(GM-CSF)(40ng/ml)and interleukin-4(IL-4)(10ng/ml)in vitro.DCs were identificated by flow cytometry detecting surface markers CD11C.The DCs were randomly divided into control group,CSE exposed group,CD40 blocking CSE exposed group and CD40 blocking control group.The CD4+T cells were selected by immunomagnetic beads from spleen of male Balb/c mice.The Dendritic cells were randomly divided into DCs co-cultured control group,CSE exposed DCs eo-cultured group,CD40 blocking CSE exposed DCs co-cultured group and CD40 blocking co-cultured control group which cultured with CD4+T cells(the ratio of DCs/CD4+T cells=1/10)in free serum medium for 24 hours.At the same time,the CD4+T cells from spleen of male Balb/c mice by MACS were randomly divided into CD4+T cells control group,CSE exposed CD4+T cells control group,CD40 blocking CSE exposed CD4+T cells control group and CD40 blocking CD4+T cells control group which had been cultured in free serum medium for 24 hours.The CSE exposed DCs co-cultured group,CD40 blocking CSE exposed DCs co-cultured group,CSE exposed CD4+T cells control group and CD40 block CSE exposed CD4+T cells control group were administrated with CSE at 1%concentration.The CD40 blocking CSE exposed DCs co-cultured group,CD40 blocking DCs co-cultured control group,CD40 blocking CSE exposed CD4+T cells control group and CD40 blocking CD4+T cells control group were administrated with purified NA/LE hamster anti-mouse CD40 at 10?g/ml concentration.The cells in each group had been harvested.The ratio of CD40 for DCs and Thl/Th2 for CD4+T cells and co-cultured celles were detected by flow cytometry,and the mRNA of CD40 for DCs and T-bet?GATA-3for CD4+T cells and eo-cultured cells were measured by fluorescence quantitative real-time PCR.The concentration of IFN-??IL-4?IL-10 and IL-12 in culturing medium cell supernatant of CD4+T cells group and co-cultured cells group was detected by liquid phase chipsResults:1.Flow cytometry detection:1)The expression of CD40 of DCs in CSE exposed group increased significantly than those in control group(all P<0.01).compared with CSE exposed group The CD40 of DCs in CD40 blocking CSE exposed group decreased significantly(P<0.05);2)compared with CD4+T cells control group,CSE exposed CD4+T cells controls and DCs co-cultured control group,The Th1(CD4+IFN-?+)in CSE exposed DCs co-cultured group increased(all P<0.05).but the Th2(CD4+IL-4+)decreased(all P<0.05),The Th1 in CD40 blocking CSE exposed DCs co-cultured group had been reduced as compared with CSE exposed DCs co-cultured group(P<0.05).but the Th2 had no significant difference(P>0.05);2.fluorescence quantitative PCR detection:1)The expression of CD40mRNA of DCs in CSE exposed group increased significantly than those in control group(all P<0.05).The CD40mRNA of DCs in CD40 blocking CSE exposed group decreased significantly than those in CSE exposed group(P<0.05).2)compared with CD4+T cells control group,CSE exposed CD4+T cells controls,DCs co-cultured control group,The T-bet mRNA in CSE exposed DCs co-cultured group were increased(all P<0.05),but the GATA-3 mRNA was decreased(all P<0.05)The T-bet mRNA in CD40 blocking CSE exposed DCs co-cultured group was lower than those of CSE exposed DCs co-cultured group(P<0.05).but the GATA-3 mRNA had no significant difference(P>0.05);3,liquid phase chip detection:compared with CD4+T cells control group,CSE exposed CD4+T controls and DCs co-cultured control group,The concentration of IFN-? and IL-12 in culturing medium of CSE exposed DCs eo-cultured group was elevated(all P<0.05),but the concentration of IL-4 and IL-10 was decrease((all P<0.05);compared with CSE exposed DCs co-cultured group,The concentration of IFN-? and IL-12 in culturing medium of CD40 blocking CSE exposed DCs co-cultured group decreased than those of(P<0.05),but the IL-4 and IL-10 had no significant difference(P>0.05)Conclusion:Cigarette smoke extract can lead to the imbalance of Thl/Th2 which by promoting dendritic cells to induce CD4+T cells differentiating into Th1 cells and inhibiting the differentiation Th2 cells.CD40-CD40L pathway plays an important role in cigarette smoke extract stimulating dendritic cells to induce CD4+T cells polarizing to Thl cells.PART ?EFFECT OF ERYTHROMYCIN ON MURINE DENDRITIC CELLS EXPOSED TO CIGARETTE SMOKE EXTRACT AND THEIR IMPACT ON INDUCING CD4+T CELLS DIFFERENTIATING TO TH1/TH2 CELLSObjective:To observe the effect of erythromycin on expression of common stimulate molecule of murine dendritic cells which exposed to cigarettes moke extract and their impact on inducing CD4+T cells polarizing to Thl/Th2 cells.Methods:About 50 SPF class male balb/c mice,aged 6 to 8 weeks,weight of 22g to 25g,dendritic cells(DCs)had been generated from bone marrow-derived mononuclear cells of these mice,by which culture in RPMI1640 medium containing 10%fetal bovine serum(FBS)with granulocyte-macrophage colony-stimulating factor(GM-CSF)(40ng/ml)and interleukin-4(IL-4)(10ng/ml)in vitro.DCs were identificated by flow cytometry detecting surface markers CD11C.The DCs were randomly divided into control group,CSE exposed group,erythromycin intervening CSE exposed group and erythromycin intervening control group.The CD4+T cells were selected by immunomagnetic beads from spleen of male Balb/c mice.The Dendritic cells were randomly divided into DCs co-cultured control group,CSE exposed DCs co-cultured group,erythromycin intervening CSE exposed DCs co-cultured group and erythromycin intervening co-cultured control group which cultured with CD4+T cells(the ratio of DCs/CD4+T cells=1/10)in free serum medium for 24 hours.At the same time,the CD4+T cells from spleen of male Balb/c mice by MACS were randomly divided into CD4+T cells control group,CSE exposed CD4+T cells control group,CD40 blocking CSE exposed CD4+T cells control group and CD40 blocking CD4+T cells control group which had been cultured in free serum medium for 24 hours.The CSE exposed DCs co-cultured group,erythromycin intervening CSE exposed DCs co-cultured group,CSE exposed CD4+T cells control group and erythromycin intervening CSE exposed CD4+T cells control group were administrated with CSE at 1%concentration.The erythromycin intervening CSE exposed group and erythromycin intervening control group were administrated with erythromycin at 100?g/ml concentration..The cells in each group had been harvested.The ratio of CD40 for DCs and Thl/Th2 for CD4+T cells and co-cultured celles were detected by flow cytometry,and the mRNA of CD40 for DCs and T-bet?GATA-3for CD4+T cells and co-cultured cells were measured by fluorescence quantitative real-time PCR.The concentration of IFN-??IL-4?IL-10 and IL-12 in culturing medium cell supernatant of CD4+T cells group and co-cultured 0ells group was detected by liquid phase chipsResults:1.Flow cytometry detection:1)The expression of CD40 of DCs in CSE exposed group increased significantly than those in control group(P<0.01).compared with CSE exposed group The CD40 of DCs in erythromycin intervening CSE exposed group decreased significantly(P<0.05);2)compared with CD4+T cells control group,CSE exposed CD4+T cells controls and DCs co-cultured control group,The Thl(CD4+IFN-T+)in CSE exposed DCs co-cultured group increased(all P<0.05).but the Th2(CD4+ IL-4+)decreased(all P<0.05),The Thl in erythromycin intervening CSE exposed DCs co-cultured group had been reduced as compared with CSE exposed DCs co-cultured group(P<0.05).but the Th2 had no significant difference(P>0.05);2.fiuorescence quantitative PCR detection:1)The expression of CD40mRNA of DCs in CSE exposed group increased significantly than those in control group(all P<0.05).The CD40mRNA of DCs in erythromycin intervening CSE exposed group decreased significantly than those in CSE exposed group(P<0.05).2)compared with CD4+T cells control group,CSE exposed CD4+T cells controls,DCs co-cultured control group,The T-bet mRNA in CSE exposed DCs co-cultured group were increased(all P<0.05),but the GATA-3 mRNA was decreased(all P<0.05)The T-bet mRNA in erythromycin intervening CSE exposed DCs co-cultured group was lower than those of CSE exposed DCs co-cultured group(P<0.05).but the GATA-3 mRNA had no significant difference(P>0.05);3,liquid phase chip detection:compared with CD4+T cells control group,CSE exposed CD4+T controls and DCs co-cultured control group,The concentration of IFN-T and IL-12 in culturing medium of CSE exposed DCs co-cultured group was elevated(all P<0.05),but the concentration of IL-4 and LL-10 was decrease((all P<0.05);compared with CSE exposed DCs co-cultured group,The concentration of IFN-?and IL-12 in culturing medium of erythromycin intervening CSE exposed DCs co-cultured group decreased than those of(P<0.05),but the IL-4 and IL-10 had no significant difference(P>0.05)Conclusions In cigarette smoke exposure condition,Erythromycin plays an important role in immune regulation,It can suppress Dendritic cells of which exposed to cigarette smoke extract to induce CD4+T cells polarizing to Th1 cells,mainly through the inhibition of CD40-CD40L signal pathway. |
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