The Effect Of STIM1/STIM2on FcγRⅡ Function Of Human Monocyte-derived Dendritic Cells

Dendritic cells （DCs） are the most powerful professional antigenpresenting cells （APCs） in vivo, which directly regulates the balance ofbetween immune response with immunotolerance. DCs two-waysregulation is closely related to their different stages of their owndevelopment, and DCs’ differentiation and maturation is inseparablewith the expression of FcγR. FcγR involved in DCs’ differentiation,maturation, and immunological effects, mainly by the activation motif（ITAM） and inhibition motif （ITIM）. Stromal interactionmolecule-1（STIM1） is a recently discovered Ca²⁺channel regulatoryprotein, called the intracellular sensor of detecting calcium content ofthe ER, which is a important molecules of capacitative calcium influxsystem, with the potential of effectively regulating the role of DCsimmune function. Stromal interaction molecule-2（STIM2） play animportant role in the later stages of Ca²⁺channels in response, regulatethe basis of Ca²⁺inflow, Ca²⁺flow not only involved in regulating thefunction of T, B and other immune cells, but also is the main elements ofITAM and ITIM play activation and suppression. The function of FcγRis caused by the realization of the flow of Ca²⁺through intracellar ITAMand ITIM, Ca²⁺flow may be an important part of that affect DCsdifferentiation and maturation. Therfore, it has important scientificsignificance to approach the role of STIM1and STIM2in regulation ofDCs’ differentiation and maturation by FcγR, and to explore the targetof the regulation of immune response in DCs. Objective：To elucidate that the effect of STIM1and STIM2in immuneregulation of DCs by exploring the role of STIM1and STIM2inmaturation of dendritic cells in regulation of the FcγR.Methods:1. The establishment of DCs culture system in vitroThe monocytes were isolated from human peripheral bloodmononuclear cells through the method of attachment to the plastic surfaceand ficoll centriufgationan. The monocytes were stimulated by GM-CSF（100ng/mL） and IL-4（100ng/mL） for6days to induce DCsdifferentiation, the cells were obtained as immature DCs. After6daysDCs were stimulated with LPS（1μg/mL） for24h to induce maturation.Inverted phase contrast microscope was used to observe the change ofDCs morphology. Flow cytometry was used for analysis DCs phenotype.2.The effect in expression of STIM1and STIM2by activating theFcγRFlow cytometry and immunofluorescence was used for analysis theexpression of DCs activating receptor FcγRⅡa and inhibitory receptorFcγRⅡb; Real-time PCR and Western-Bolt were used for analysis theexpression of STIM1and STIM2in immature dendritic cells and maturedendritic cells; IV.3and7.3respectively combined with F（ab’）₂as IC,Real-time PCR and Western-Bolt were used for analysis the expression ofSTIM1and STIM2when IC cross-linking FcγRⅡa and FcγRⅡb for3minutes and30minutes.3.The establishment and identification of the RNA interferencesystem of STIM1and STIM2By chemical method to synthesized siRNA and to deliver siRNAinto DCs by Lipo chemical transfection method, Real-time PCR to detectthe effect of transfection, MTT assay transfection cell activity after24 hour.4. The effect of DCs maturation by regulate the expression ofSTIM1and STIM2Flow cytometry was used for phenotypic analysis of DCs whenDCs were stimulated by LPS or cross–linked activating receptor FcγRⅡaafter STIM1and STIM2were silencing by siRNA technology.Results:1. The immature DCs moderate express inhibitory receptor FcγRⅡb（57±7.1）%, FcγRⅡb moderately lower when DCs were inducedmaturation with LPS （22±2.1）%, but the expression of FcγRⅡa nosignificant change （43.9±4.3,48±2.8）%.2. The immature DCs high express STIM2, however STIM2werelower express in mature dendritic cells （P<0.05）. the expression ofSTIM1were no significant change with DCs mature. the expression ofSTIM1and STIM2availably inhibited by siRNA technology, theinhibition rates were70.1%and65.8%. and it is no significant effect onthe activity of DCs, the survival rate is （94.17±12.38）%and（97.17±5.07）%. The dendritic cell mature status no significant changewhen STIM1and STIM2were silencing by siRNA technology, DCs isstill high expression of CD80,CD86,CD40,CD83and HLA-DR（P>0.05）.3. The expression of STIM1and STIM2no significant changewhen IC cross-link activating and inhibitory FcγRⅡ for3minutes.When cross-linking7.3for30minutes, the expression of STIM1andSTIM2conspicuous increased （p<0.01）. However, the expression ofSTIM1and STIM2conspicuous decreased when cross-linking IV.3for30minutes （p<0.01）. Whether individually or jointly silencing STIM1and STIM2, activate FcγRⅡa may also lead to a especialy maturephenotype in DCs, DCs is high expression of CD80, CD40andHLA-DR, moderate express CD86, but the expression of CD83were lower.Conclusion:1. DCs express both STIM1and STIM2, and the main expressionof STIM2in iDCs. the expression of STIM1no significant change withDCs mature.2. DCs can still be induced to mature by LPS when STIM1andSTIM2were inhibited.3. The activation of FcγRⅡis closely related to the expression ofSTIM1and STIM2，upregulation of STIM1and STIM2when activingFcγRⅡb；downregulation of STIM1and STIM2when activing FcγRⅡa.4. FcγRⅡa can induce relatively mature phenotype of DCs，butindependent on the expression of STIM1and STIM2.