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Abstract

Besides malignant cells, tumours are composed of vascular, hematopoietic and mesenchymal cells, amongst which in particular macrophages can be found numerously within the tumour infiltrate. So called tumour-associated macrophages (TAM) exert important tumour-supportive roles and are involved in tumour initiation, progression and metastasis as providers of angiogenic, anti-inflammatory and matrix remodelling factors. In numerous different cancers, high TAM density correlates with poor patient prognosis. The plasticity of gene regulation allows macrophages to adapt their phenotype to local requirements. Therefore TAM populations are highly heterogeneous and adapt to the corresponding microenvironment of the particular tumour region. The identification of further TAM macrophage markers is indispensable regarding the necessity of functional characterization of distinct TAM subsets. In the past years, our group has identified Ms4a8a, Stabilin-1 and Lyve-1 as novel markers of murine TAM. In vitro, Lyve-1 expression could be induced in murine bone marrow derived macrophages by the stimulation with tumour conditioned medium, dexamethasone and IL-4. In this thesis the main aim was to comprehensively characterize expression and processing of Lyve-1 in macrophages in vitro and in vivo. The type I transmembrane glycoprotein Lyve-1 is part of the link domain superfamily with 43 % sequence homology to the hyaluronan receptor CD44. Lyve-1 has first been identified as a lymphatic endothelial cell specific HA receptor which also binds different growth factors like FGF-2 and VEGF. Lyve-1 is involved in lymphangiogenesis in endothelial cells. By immunohistological analyses we could demonstrate the presence of Lyve-1+ macrophages in human melanoma specimen and in murine B16 transplant tumours. In human peripheral blood mononuclear cells (pBMC), which were isolated from buffy coats, seven days of stimulation with M-CSF / dexa / IL-4 (MDI) led to the induction of Lyve-1 in 25 % of the stimulated cells. Expression analysis of combinations of macrophage marker revealed that Lyve-1+ pBMC were more oriented towards a M2-like phenotype. In addition, we found that the induction of Lyve-1 in vitro is crucially dependent on the activation of the glucocorticoid receptor (GR) and can be impaired by inhibition of the p 38 MAPK signalling cascade. In vivo, the activation of GR seems to play a secondary role for Lyve-1 induction, as Lyve-1+ macrophages were detected in murine B16F10 tumours derived from mice with a myeloid specific deletion of the GR. Functional analyses were accomplished using genetically engineered Lyve-1 overexpressing cell lines. In the human macrophage like cell line U937 Lyve-1 XIV was expressed in a highly glycosylated state. Even though binding to HA could be demonstrated, ligand binding did not induce proliferation as it has been reported for Lyve-1+ endothelial cells. In an adhesion assay we identified fibronectin as a further binding partner of Lyve-1. In analogy to CD44, which is shedded from the cell surface by proteolytic cleavage, we supply evidence for the existence of a soluble form of Lyve-1 in the cell culture supernatant of transgenic U937. Soluble Lyve-1 (sLyve-1) is produced by a metalloproteinase mediated shedding process in macrophages and endothelial cells in a comparable fashion. To address our hypothesis that sLyve-1 might function as a decoy receptor, we incubated melanoma cell lines with macrophage derived and synthetic sLyve-1 and observed reduced tumour cell proliferation. Physiological relevance of Lyve-1 shedding was demonstrated by the quantification of sLyve-1 in human plasma. The concentration of sLyve-1 was significantly increased in plasma samples derived from psoriasis patients. To examine the relevance of Lyve-1+ macrophages for tumour growth we injected B16 melanoma tumour cells subcutaneously into the flank of Lyve-1-/- mice. After ten days enhanced tumour growth due to increased tumour cell proliferation was observed in the knockout mice in comparison to wild type controls. The exact contribution of Lyve-1+ macrophages to this effect still needs further clarification. Therefore, we could demonstrate that Lyve-1 is a marker of a M2-like macrophage subpopulation in human and murine melanoma. By proteolytic cleavage of the ectodomain the function of Lyve-1 is modulated and thus plays also an important role for tumour growth and progression.