Endothelial Cell-Derived Microparticules (EMPs) Can Induce In Vitro Maturation of Both Plasmocytoid and Myeloid Dendritic Cells.

The implication of plasmocytoid dendritic cells (pDCs) in transplantation is more and more reported, especially because of their tolerogenic properties supported by the expansion of regulatory T cells. Endothelial microparticles (EMPs) are plasma membrane-derived vesicules shed from endothelial stimulation involved in physiological and pathological processes, including intercellular communication, hemostasis and immunity. However, the interaction between DC and EMP are not well-known, especially in the context of alloreactivity. Therefore, the aims of the current study were to explore the DC/EMP interactions, more specifically the capacity of EMPs to modulate the maturation of DC. Firstly, we validated an original co-culture model involving a tumoral pDC lineage (GEN 2.2) and EMPs generated in a standard fashion from an endothelial cell lineage (HMEC U608), stimulated or not with TNF-α . EMPs was analyzed by FACS (FITC labelled annexin V) and supernatant resulting from the last wash of EMPs was used as negative control. By incubating increasing amounts of EMPs we showed that EMPs can lead to GEN pDC maturation in a dose- effect manner as evidenced by a significative increase in the HLA-DR and CD83 expression, compared to the negative controls or, of importance, compared to the maturation with CPG-A (ligand of TLR9 mimicking bacterial DNA), CD40L or R848 (ligand of TLR7 mimicking viral RNA). To confirm and expand these findings, we then cocultured with EMPs (and similar controls)

1. circulating pDC obtained by an anti-BDCA-4 MoAb-mediated positive selection of healthy donors PBMC , and

2. myeloid DC (mDC) obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4.

Maturation of both normal circulating pDC and mDC occurred in the presence of EMPs and once again, in a dose-dependant fashion. The cytokine secretion pattern of the EMPs- matured mDC and pDC, determined by the Luminex technology, was as expected for such cell populations. The EMPs matured pDC secreted IL-1, IL-6, IFN-α , IL-12 and IL-10 but not TGF-β . In contrast, the EMPs matured mDC secreted mainly proinflammatory cytokines : IL-1, IL-6 and IL-8 but not IL-10. Overall, our studies demonstrate that EMPs can induce the maturation of both pDC and mDC. Such endothelial microparticules, generated by inflammatory and hemostatic disorders, could play a role in post-transplant immune responses. Determining the factors resulting in EMPs-induced pDC vs mDC maturation may open the possibility for EMPs-related immuno-intervention.