TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/26811/
A1  - Kloss, Loreen
N2  - Macrophages are phagocytic cells of the innate immune system that exert important functions for immunological and homeostatic processes. They have a high plasticity and can quickly adapt to different environments. While Th1 cytokines induce a pro-inflammatory macrophage phenotype (M1), Th2 cytokines promote an anti-inflammatory one (M2). At tumor initiation a M1 phenotype is predominant, which then switches to a M2-like phenotype during tumor progression. These tumor-associated macrophages (TAM) promote tumor growth and metastasis by the secretion of growth factors, matrix metalloproteinases and immunosuppressive mediators such as arginase-1, IL 10 and TGF-ß. In many tumor entities including melanoma a high TAM infiltration correlates with a poor patient outcome. Thus, macrophages provide a promising therapeutic target for adjuvant tumor therapies. In the past our group identified several novel markers for TAM such as STABILIN-1 and LYVE-1. These markers can be induced in vitro by the treatment of peripheral blood monocytes (pBM) with M-CSF/dexamethasone/IL-4 (MDI). Microarray analysis of these in vitro generated M2-like macrophages identified the purinergic, G protein coupled receptor P2Y12 as the highest up-regulated gene. So far, P2Y12 has been known to be expressed on microglia and platelets and is targeted therapeutically with clopidogrel, ticagrelor and prasugrel to treat thrombotic disorders. Since the expression of P2Y12 in TAM has not been described yet, the aim of this study was to characterize P2Y12 expression and its function in M2-like macrophages in vitro as well as in TAM in vivo. Since all commercially available antibodies failed to detect P2Y12 in transgenic P2Y12+ U937 cells, we generated a peptide antibody against human P2Y12 and confirmed the expression of P2Y12 receptor in MDI-treated pBM as well as in CD68+ and CD63+ TAM of melanoma in situ. Like in the human system, P2y12 can also be induced by the treatment of murine bone marrow-derived macrophages (BMDM) with MDI. We detected P2y12 expression in CD11b+ cells isolated from spleen, peritoneal fluid and subcutaneously transplanted B16F10 tumors of tumor-bearing mice. P2Y12 expression in murine TAM was confirmed by immunohistochemical staining of B16F10 tumors. Treatment of murine and human transgenic P2Y12+ macrophage-like cell lines with the P2Y12 receptor agonist ADP increased the expression of several cytokines and chemokines (in the murine system CXCL2 and TNF-? and in the human system CXCL2, CXCL7 and IL-8), indicating an immunomodulatory role for the receptor. Analyzing signaling pathways underlying the induction of these cytokines, we detected ERK as well as Akt phosphorylation in ADP-treated P2Y12+ U937 cells. Inhibition of these signaling pathways significantly impaired CXCL2, CXCL7 and IL-8 secretion. Since ADP acts as a chemoattractant for microglia cells, the migratory potential of P2Y12+ macrophages was evaluated. We could show that ADP promotes the migration of human pBM(MDI) as well as murine P2Y12+ Raw 264.7 cells in a transwell chamber. Pre-treatment with the P2Y12 antagonist PSB0739 abrogated the ADP-induced chemotaxis of P2Y12+ cells. Since it is known that dying cells release nucleotides that act as find-me signals for phagocytic cells, we set up a co-culture experiment with P2Y12+ Raw 264.7 cells and B16F1 melanoma cells, which we pre-treated with puromycin to induce cell death. Dying tumor cells promoted the migration of P2Y12+ macrophages. Administration of the ATP/ADP cleaving enzyme apyrase to the dying tumor cells or treatment of P2Y12+ Raw 264.7 cells with PSB0739 abrogated the cell-death induced migration of P2Y12+ macrophages. Taken together, our results indicate that P2Y12 is an important immunomodulatory macrophage receptor, which triggers migration of these cells towards ADP-rich tumor areas and is able to modulate the inflammatory environment upon ADP activation.
TI  - Molecular and Functional Characterization of P2Y12 Expression in Tumor-associated Macrophages
Y1  - 2019///
AV  - public
ID  - heidok26811
ER  -