Lympathic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1⁺, F4/80⁺, CD11b⁺ macropahages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: Implications for the assessment of lymphangiogenesis

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specitic hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b⁺, F4/80⁺ tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1⁺ macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVF-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/ dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1⁺, stabilin-1⁺ double-positive, while 9.9% were LYVE-1⁺, stabilin-1^- and 33.5% were LYVE-1^-, stabilin-1⁺. Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1⁺, stabilin-1⁺ non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1⁺ lymphatics and LYVE-I⁺ tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.