No abstract

IntroductionThe interactions between the immune system and angiogenesis are determinant for the regulation of tumor growth. The mechanisms of these interactions are not well understood at this time. Dendritic cells (DCs) are known to play a central role in these interactions by releasing vascular endothelial growth factor-A (VEGF-A). 1,2 VEGF-A is able to induce endothelial-like differentiation of tumor-infiltrating precursors of DCs and their migration to vessels to participate to vasculogenesis. 3 VEGF-A was also reported to inhibit the function of mature DCs. 4 Immune cells such as tumor-associated macrophages and T cells were also reported to regulate angiogenesis through the secretion of potent angiogenic mediators such as angiopoietin-2 and interleukin-17 . 5,6 We demonstrated previously that ATP up-regulates the expression by monocyte-derived DCs (MoDCs) of numerous genes that may play a role in immunosuppression, in particular thrombospondin-1 and indoleamine 2,3-dioxygenase. 2,7 Some epidermal growth factor receptor (EGFR) ligands were also ATP target genes in MoDCs, in particular, amphiregulin (AREG), which was the most highly up-regulated gene. AREG is a cell type-dependent mitogenic factor that binds to ErbB1 receptor, also called EGFR. 8 AREG is implicated in tumorigenesis and angiogenesis. 9,10 Ma et al showed a reduction of the tumor mass and the intratumoral vascularization using an antisense cDNA of AREG in a breast cell line. 11 A clinical study showed that there was a significant overexpression of AREG and VEGF in primary breast cancer. 12 Kato et al showed that AREG can induce the proliferation of rat vascular smooth muscle cells and could be implicated in arterial remodeling. 13 We demonstrated here for the first time that human and mouse DCs are a source of AREG, an EGFR ligand with tumorigenic properties. Methods MiceMale 6-to 8-week-old C57BL/6 mice were obtained from Charles River Laboratories. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health (NIH) and were approved by the Comission d'Ethique et du Bien-Etre Animal (CEBEA) ethical committee. ReagentsATP, adenosine 5Ј-O-(3-thiotriphosphate; ATP␥S), UTP, prostaglandin E2 (PGE 2 ), adenosine (Ado), NECA (5Ј-(N-ethylcarboxamido)adenosine), suramin, MRS1754, and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich. Monoclonal anti-human and anti-mouse AREG blocking antibodies were purchased at R&D Systems. Preparation of human monocyte-derived DCsPeripheral blood mononuclear cells were isolated from leukocyte-enriched buffy coats of healthy volunteer donors by standard density gradient centrifugation using Lymphoprep solution from Nycomed. Mononuclear cells (2.5 ϫ 10 8 ) were allowed to adhere during 1 hour and 30 minutes at 37°C at 5% CO 2 in 75-cm 2 cell culture flasks. Nonadherent cells were removed, and adherent cells were cultured in 15 mL of culture medium For personal use only. on April 4, 2019. by guest ...

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPγS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPγS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPγS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPγS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPγS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPγS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPγS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPγS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.