| Objectives:? To detect the level of serum transglutaminase specific IgE in AD patients and analyze its correlation with the disease severity.? To detect the mRNA expression levels of TGMs and protein levels of TGM3 in the epidermal lesions of AD patients.? To detect the mRNA expression levels of TGMs in human immortalized keratino-cytes(HaCat cells)under different inflammatory conditions.? To detect the interference efficiency of CD209siRNA to CD209,and detect the bind-ing ability of CD209 with TGM3.? To detect the activation status of monocyte-derived dendritic cell(MDDC)after stimulated by TGM3.?Study the differentiation and polarization of naive T cells induced by TGM3-MDDCs.Methods:? Using immunocapture technology and biotinylated enzyme immunoassay method,we detected the expression level of serum TGMs sIgE in 77 cases of AD patients(including 44 adolescents and adults,33 infants and children),40 cases of adult patients with psori-asis vulgaris and 30 cases of adult healthy controls,and analyzed the correlation of TGMs sIgE level with the disease severity of AD.? qRT-PCR was used to assay the mRNA expression levels of TGM1,TGM2,TGM3 and TGM5 gene in epidermal lesions.Laser scanning confocal microscopy was applied to detect the protein expression level of TGM3 at the same time.? qRT-PCR was used to detect the mRNA expression levels of TGM1,TGM2,TGM3 and TGM5 in cultured HaCat cells with the stimulations of Derp,TSLP,and IL-1? re-spectively.? qRT-PCR and flow cytometry(FCM)were separately used to detect the mRNA and protein expression levels of CD209 after interfered by CD209 siRNA.ELI SA,Laser confocal microscopy and FCM were used to detect the binding ability of TGM3 with CD209.EGFP-CD209 transfected 293T cells and MDDCs were used as models.? FCM was used to detect the expression levels of CD80,CD83,HLA-DR and CD86 on TGM3-MDDCs.Cytometric Bead Array was carried out to detect the expression levels of proinflammatory cytokines included IL-1?,IL-6,IL-8,IL-10,IL-12 and TNF-? in cell supernatants.? Naive T cells were co-cultured with TGM3-MDDCs for 5 days firstly,and were stimulated with TGM3 for another 24h.FCM was used to detect the polarization of naive T cells.IL-4,IL-12,TNF-?,IL-17A,IL-10,IFN-?,IL-6 and IL-17A in cell supernatants were detected by the method of CBA.Results:? The average OD value of TGMs sIgE in adolescent and adult AD patients and adult psoriasis vulgaris were higher than that in adult healthy controls(t=7.38 and 4.83,P<0.001).The average value of TGMs sIgE in intrinsic AD group was higher than that in extrinsic AD group(t=2.27,P=0.02).There were no correlation between TGMs sIgE level and disease related indexes,such as age,disease duration,SCORAD score,eosino-phil counts and serum total IgE in AD patients(t=0.03,0.14,-0.04,-0.08 and 0.06,P=0.78,0.24,0.74,0.54 and 0.62).? The mRNA expression levels of TGM3 and TGM5 in epidermal lesions of AD group were significantly higher than those in healthy controls(P=0.001 and 0.01).The protein expression of TGM3 in epidermal lesions of patients with AD was higher than that in normal skins.? Compared with the blank control group,TGM1 expression level was increased sig-nificantly in Derp lOug/ml and TSLP 20ng/ml stimulation group for 12h,24h and 36h(P values<0.01),TGM1 expression level was also increased significantly in IL-1?lOng/ml and IL-1? 50ng/ml stimulation group for 24h.Compared with the blank control group,TGM2 expression level was increased significantly in Derp lOug/ml stimulation group for 12h and 24h,TLSP 50ng/ml and IL-1? 50ng/ml stimulation group for 24h and 36h,and IL-1? 50ng/ml stimulation group for 24h(P values<0.001).Compared with the blank control group,TGM3 expression level was increased significantly in TLSP 20ng/ml stimulation group for 12h,derp lOug/ml stimulation group for 24h and 36h,TLSP 50ng/ml and IL-1? 50ng/ml stimulation group for 24h(P values<0.01).Compared with the blank control group,TGM5 expression level was increased significantly in derp lOug/ml stimulation group for 24h and 36h,TSLP 50ng/ml stimulation group for 24 h,and TLSP 20ng/ml stimulation group for 36h(P values<0.001).?The mRNA expression of CD209 was decreased significantly after treated with CD209siRNA for 24 hours.The protein expression of CD209 was decreased to the maximum with the interference for 72 hours.TGM3 could bind with CD209 by ELISA assay.TGM3 can be recognized,up-taken and engulfed into cells by CD209 on the sur-face of 293T cells and MDDCs,and could be blocked by CD209 blocking antibody and CD209 siRNA partially.? Compared with several control groups and CD209 blocked(a-CD209)group,the ex-pression levels of CD83,CD86 on the cells' surfaces and IL-6 in cell supernatants were significantly increased by stimulated with TGM3(CD83:P<0.001;CD86:P=0.007;IL-6:P=0.006).? Compared with the controls,the expression level of IFN-y in the TGM3-MDDC-T cells was higher.Also,TNF-a and IFN-y expression levels in conculture supernatants were increased than those in control groups(P=0.0018).Conclusions:? TGMs sIgE levels in serum of patients with AD were significantly increased.TGMs might be one of major auto-antigens and intrinsic antigens of AD.? The mRNA and protein expression levels of TGM3 in the epidermal lesions of AD were increased.? The mRNA expression levels of TGM1,TGM2,TGM3 and TGM5 in Hacat cells were elevated under the stimulations with Derp,TSLP and IL-1?.? CD209 siRNA could partially interfere the expression of CD209.TGM3 can be rec-ognized,uptaken and engulfed into dendritic cells as an auto-antigen by CD209.? TGM3 can stimulate MDDCs activation and induce higher level expression of IL-6.? TGM3-MDDCs may polarize T cells to differentiate into Thl cell subsets. |
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