Neutrophil-derived microparticles (MP) have become important mediators of inflammation, coagulation and vascular homeostasis. MP carry surface antigens, proteins and adhesion molecules from their originating cell and can mediate intercellular cross-talk. Human neutrophil contain high levels of the anti-inflammatory protein annexin 1 (AnxA1), which contributes to mechanisms activated in the host to keep under control cell activation and trafficking in the resolution of inflammation. We investigated the role of MP released by all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemic (APL; NB4) cells during the process of granulocytic differentiation.
We determined the expression of AnxA1 and its receptor (FPRL1) on the NB4 cells and their MP by flowcytometry, real-time PCR and western blotting. The anti-inflammatory effect of AnxA1was determined by the transmigration assay.
AnxA1 was constitutively expressed on the surface of ATRA-untreated NB4 cells. ATRA treatment of NB4 cells can significantly enhance their surface expression of Anx-A1 in a time dependent manner, but did not change their mRNA expression or release of free AnxA1 into the conditioning medium. We further determined the amount of MP in the CM by flowcytometry. Significantly higher number of MP was released by the ATRA-NB4 cells, as compared with those by ATRA-untreated NB4 cells (p<0.05). Further study demonstrated that AnxA1was expressed on the surface of MP, and ATRA can also enhance the release of Anx-A1 (+) MP from ATRA-NB4 cells. Transmigration studies indicated that exogenous Anx-A1 protein can inhibit the transmigration of ATRA-NB4 cells in a time- and dose-dependent manner (p<0.05 & p<0.05; respectively). AnxA1 receptor (FPRL-1) was expressed on the surface of both ATRA-treated and ATRA-untreated NB4 cells. Blocking the FPRL1 on the surface of ATRA-NB4 cells with its specific antibody can further enhance their transmigration activity, indicating that the important anti-migratory role of AnxA1-FPRL1 axis on the ATRA-treated NB4 cells. We further demonstrated that ATRA-treated NB4 cells-derived MP can also significantly inhibit the transmigration of ATRA-NB4 cells (p<0.05). Similarly, anti-migratory effect of MP can be attenuated when their surface Anx-A1 was blocked with its specific antibody, implying that AnxA1 mediates the rapid anti-migratory effects of MP. We further determined the mode of action that Anx-A1(+) MP acted on the recipient cells. MPs were pre-labeled with FITC-conjugated Anx-A1 monoclonal antibody before incubating with recipient ATRA-treated NB4 cells. Flowcytometry demonstrated that FITC-labeled Anx-A1 was detected on 87% of recipient ATRA-treated NB4 cells, indicating that Anx-A1 (+) MP can fuse into the surface of the recipient ATRA-treated NB4 cells.
differentiated APL cells-derived microparticles contain functionally active AnxA1 that confers them anti-migratory properties which contributes to the control mechanism of cell recruitment.
No relevant conflicts of interest to declare.
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