title: New model of macrophage acquisition of the lymphatic endothelial phenotype.
abstract: BACKGROUND
  Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions.
  
  
  RESULTS
  Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b(+)/VEGFR-3(+) LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels.
  
  
  CONCLUSIONS
  We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the "RAW model" mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.
authors.affiliation: Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Hall
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Volk-Draper
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Flister
authors.forename: Sophia
authors.initials: S
authors.lastname: Ran
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000096:biosynthesis; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D008208:Lymphatic System / Q000166:cytology*; D008264:Macrophages / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000378:metabolism
chemical_list: D039481:CD11b Antigen; D008070:Lipopolysaccharides; D016328:NF-kappa B; D049452:Green Fluorescent Proteins; D040321:Vascular Endothelial Growth Factor Receptor-3