The Involvement Of ANXA1 In The Reversion Of Maturation Of MoDCs By α-Melanocyte Stimulating Hormone

Dendritic cells (DCs), which are potent professional antigen presenting cells (APCs), play the unique role in immune system because of their abilities to activate na?ve T cells and initiate a primary immune response. The maturation/activation state of DCs might be the control point for the induction of peripheral tolerance. Mature DCs (mDCs) are potent APCs enhancing immune response, whereas immature DCs (iDCs) are involved in the induction of immune tolerance. The study on the differentiation and maturation of DCs will contribute to the elucidation of the mechanisms of autoimmune disease, transplant rejection and anti-tumor immunity and the exploration of new therapy.A bidirectional regulation exists between the neuroendocrine and immune systems, through which the neuroendocrine system signals the immune system via hormonal and neuronal pathways, and the immune system signals the neuroendocrine system through cytokines and other immune molecules. This delicate regulation net plays an important role in resisting disease and maintaining homeostasis.The neuropeptide,α-melanocyte-stimulating hormone (α-MSH) is well known for its anti-inflammatory and immunomodulatory capabilities. The monocyte-derived dendritic cell ( MoDCs ) express MC-1R, which is the receptor ofα-MSH. Moreover, the expression of the costimulatory molecules CD86 and CD40 was down-regulated on MoDCs in the presence ofα-MSH. The proteomics analysis indicated that the increased expression of ANXA1 may be involved in the reversion of maturation of TNF-α-MoDCs byα-MSH. The result of Western-Blot also showed that compared with TNF-α-MoDCs, an up-regulation of ANXA1 was in the protein extraction from TNF-α+α-MSH-MoDCs.ANXA1, formerly known as lipocortin 1, is a 37kDa member of the annexin superfamily of Ca2+ and phospholipid binding proteins. ANXA1was first identified as a glucocorticoid (GC)-inducible protein in rat peritoneal macrophages and was heralded as a potential mediator of the powerful anti-inflammatory actions of these steroid hormones. Subsequent studies have confirmed that ANXA1 does indeed contribute to the anti-inflammatory reactions of GCs. These studies have also revealed that the protein fulfills wider brief as in intra- and intercellular signaling molecule and that it is concerned with processes as diverse as cell growth and differentiation, apoptosis, vesicle fusion, endocytosis, exocytosis, and neuroprotection.Our previous works demonstrate that ANXA1 is decreased expression in TNF-α-MoDCs, while ANXA1 is increased expression during the reversion of maturation of TNF-α-MoDCs byα-MSH. However, although ANXA1 has been reported to function as an anti-inflammatory factor, its role on DC maturation has not been examined. In this study, the ANXA1 over-expressing plasmid was transfected into TNF-α-MoDCs to complement the decreased expression of ANXA1. On the other hand, small-interfering RNA targeted against ANXA1 was used in TNF-α+NDP-MSH-MoDCs. [Nle4-D-Phe7]-α-MSH, also called NDP-MSH, is one of the several potent analogues ofα-MSH. The expression of ANXA1 is also up-regulated in TNF-α+ NDP-MSH-MoDCs.To determine the effects of ANXA1 on the MoDCs maturation, we compared different groups of MoDCs in terms of surface markers, endocytic capacity, cytokine production, and the ability to stimulate T cells. The results are as follows.In the first part of our experiments, the ANXA1 over-expression plasmid was constructed by a series of molecular cloning technologies. Compared with TNF-α+pShuttle-CMV-MoDCs, the expression of HLA-DR was increased on TNF-α+pShuttle-CMV-ANXA1-MoDCs. Up-regulated IL-12 secretion and down-regulated IL-10 were also observed. The endocytic activity of TNF-α+pShuttle-CMV-ANXA1-MoDCs was significantly decreased. The ability of pShuttle-CMV-ANXA1-MoDCs to stimulate allogeneic T cell was enhanced. The results are not consistent with our hypothesis that over-expression of ANXA1 could reverse the maturation of MoDCs for the anti-inflammatory property of ANXA1. But our results are similar with that reported by Tomoko Smyth. It was found that in contrast to exogenous administration of N-terminal peptides of ANXA1 which reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. It is supposed that the over-expression of ANXA1 could not completely stimulate the signaling pathway induced byα-MSH in the reversion of maturation of TNF-α-MoDCs, so we did observe the maturation but not immature state caused by over-expression of this molecule. And there may be another explanation for the results, that proper amount of ANXA1 maintains the immature state of MoDCs, whereas excessus expression of ANXA1 may result in the maturation of MoDCs.In the second part of our experiments, ANXA1-siRNA duplexes were obtained and transfected to TNF-α+NDP-MSH-MoDCs using GeneporterⅡ. Compared with TNF-α+NDP-MSH+non-siRNA-MoDCs, TNF-α+NDP-MSH-ANXA1-siRNA-Mo DCs were significantly increased in the expression of HLA-DR, decreased in the expression of CD1a (in this experimental system, the MoDCs maturation is associated with the down-regulated CD1a, which is consisted with the result reported by Xiaochun Cao). Up-regulated IL-12 secretion and down-regulated IL-10 were also observed. The endocytic activity of TNF-α+NDP-MSH-ANXA1-siRNA-MoDCs was significantly reduced. The ability of TNF-α+NDP-MSH-ANXA1-siRNA-MoDCs to stimulate allogeneic T cells was enhanced.These data suggest that ANXA1 exerts a role in the maturation of MoDCs and is involved in the reversion of the maturation of TNF-α-MoDCs byα-MSH/NDP-MSH. Therefore, a new experimental proof is provided by this sdudy for the illumination of anti-inflammatory and immunoregulatory mechanisims ofα-MSH and the elucidation of regulatory mechanisims in maturation and function of DCs.