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Updated: May 24, 2026
Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
New model of macrophage acquisition of the lymphatic endothelial phenotype
Kelly L Hall1, Lisa D Volk-Draper, Michael J Flister
1Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
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.
Methodology/Principal Findings:
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/Significance:
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.
Insights
Researchers developed a new cell culture model using lipopolysaccharide (LPS) to study macrophage-derived lymphatic endothelial cell progenitors (M-LECPs). This "RAW model" allows detailed investigation of M-LECP differentiation and lymphatic biology.
Area of Science:
- Cell Biology
- Immunology
- Vascular Biology
Background:
- Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) are crucial for new lymphatic vessel formation.
- Understanding M-LECP differentiation, recruitment, and function is limited by their low in vivo frequency and lack of suitable in vitro models.
- Existing models hinder in-depth molecular analyses of M-LECPs.
Purpose of the Study:
- To establish a novel cell culture model for characterizing inflammation-induced macrophage-to-LECP differentiation.
- To investigate the molecular mechanisms regulating M-LECP development under controlled conditions.
- To facilitate detailed molecular analyses of M-LECPs.
Main Methods:
- Lipopolysaccharide (LPS) was used to activate RAW264.7 macrophages in vitro.
- Time-course analysis of mouse diaphragms following LPS treatment.
- Analysis of macrophage and M-LECP markers, including VEGFR-3 and VEGF-C expression.
- Co-expression analysis of lymphatic-specific genes in activated macrophages.
- In vivo studies using GFP-tagged RAW264.7 cells injected into LPS-treated mice.
Main Results:
- LPS treatment induced rapid macrophage mobilization and differentiation into M-LECPs, with approximately 50% incorporation into inflamed lymphatic vasculature.
- Activated macrophages exhibited transient VEGFR-3 expression during transition to LECPs, alongside co-expression of VEGF-C, suggesting an autocrine signaling loop.
- Injected GFP-tagged RAW264.7 cells infiltrated inflamed diaphragms and integrated into lymphatic vessels.
- Activated RAW264.7 cells showed significant overlap in lymphatic-specific gene expression with endogenous M-LECPs.
Conclusions:
- A new 'RAW model' using LPS-activated RAW264.7 cells effectively mimics key features of endogenous M-LECP differentiation.
- This model overcomes limitations of cell number and heterogeneity, enabling genetic manipulation for experimental purposes.
- The 'RAW model' provides a valuable tool for advancing research in M-LECP biology and lymphatic vessel formation.
