Effects Of STIM1/STIM2on Phenotype And Immune Function Of Human Monocyte-derived Dendritic Cells

Objective: To study the effects of STIM1/STIM2on thefunction status of dendritic cells (DCs) differentiated from human monocytes invitro, encompassing morphology, cell surface phenotype, capacity ofendocytosis and stimulation of autologous leukomonocyte. Methods:Mononuclear cells were isolated from fresh human peripheral blood by densitygradient centrifugation and successive adherence, which were derived into DCsby cytokine GM-CSF, IL-4and TNF-α induction. Then, STIM1smallinterfering RNA (siRNA), STIM2siRNA or scrambled siRNA (sc siRNA) weretransfected into DCs by liposome2000. Normal control group, DCs-iSTIM1group and DCs-iSTIM2group were set. Subsequently,(1) the morphology ofDCs were identified by inverted phase contrast microscope, transmissionelectron microscope (TEM) and scanning electron microscope (SEM);(2) DCswere immunostained for cell surface markers (including HLA-DR, CD1a,CD83, CD80, CD86, CD40), the positive expression rates and mean fluorescentintensity (MFI) were analyzed by flow cytometry (FCM) with a directimmunofluorescence method;(3) DCs were incubated with FITC-dextran, thenFCM was utilized to identify the percentage that double positive cells occupiesin PE-CD11c positive cells, by which the endocytosis ability of DCs wasevaluated;(4) allogeneic lymphocyte proliferation assay combined with MTTcolorimetric assay was applied to assess the capacity of DCs for stimulation of lymphocytes. Results:(1) Compared with normal control group, DCs inDCs-iSTIM1group and DCs-iSTIM2group have more, thinner and longersynapses, even shaped like a reticulation from TEM observation. Furthermore,it showed increasing swollen mitochondrias. However, little or no change in themorphology was maintained under SEM and inverted phase contrastmicroscope.(2) Interfered with STIM1or STIM2of DCs resulted in asignificant upregulation of cell surface phenotypes including CD80(P<0.01),CD83(P<0.01), CD86(P<0.01) and CD40(P<0.05), but little change inexpression of CD1a and HLA-DR. However, the MFI of HLA-DR and CD86were increased (P<0.05).(3) Interfered with STIM1or STIM2of DCsproductively affect DCs by a significant decrease of endocytosis capacity(P<0.05).(4) Moreover, interfered with STIM1or STIM2of DCs promotedtheir capacity for stimulation of lymphocytes, especially when DCs andlymphocytes at a ratio of1:5and1:1(P<0.01). Conclusions:(1)STIM1/STIM2defect modified the morphology of DCs. Interfere with STIM1or STIM2of DCs induced more, thinner and longer synapses, which may resultfrom that STIM1/STIM2defect entitled DCs to advanced maturation.Furthermore, interfere with STIM1lead to more swollen mitochondrias, whichmay be relevant to strengthened metabolic activity or increased programmedcell death.(2) STIM1/STIM2defect facilitate DCs maturation. Interfere withSTIM1or STIM2of DCs leads to upstream expression of the functionallyimportant cell surface phenotype of CD83, compromises their ability of endocytosis; in addition, interfere with STIM1or STIM2results in upstreamexpression of CD80, CD86and CD40, enhanced ability for stimulation oflymphocytes, which subsequently promote activation of T cells and B cells.These changes were consistent with DCs advanced maturation.(3)STIM1/STIM2defect productively interfere with DCs by modulating theirphenotype and immune function, promote maturation, and even may play anessential role in pathogenesis and mechanisms of AID. Thus, regulation ofSTIM1or STIM2may induce immunologic tolerance, which may well presenta promising view for the prevention and treatment of AID.