Temporal phenotypic features distinguish polarized macrophages in vitro

Autoimmunity. 2015 May;48(3):161-76. doi: 10.3109/08916934.2015.1027816. Epub 2015 Mar 31.


Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ + LPS (M1), IL-4 (M2a) or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5-3 h), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3-6 h) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fizz1 and Ym1) were progressively (3-24 h) increased post-stimulation. In addition, novel patterns were observed. First, and unexpectedly, cellular pro-inflammatory chemokines, MCP-1 and MCP-3 but not MCP-5, were comparably increased in M1 and M2a macrophages. Second, Vegfr1 mRNA was decreased in M1 and increased in M2a macrophages. Finally, VEGF-A was increased in the CM of M1 cultures and strikingly reduced in M2a coinciding with increased Vegfr1 expression, suggesting decreased VEGF-A in M2a CM was secondary to increased soluble VEGFR1. In conclusion, macrophage cytokine production and marker expression were temporally regulated and relative levels compared across polarizing conditions were highly dependent upon the timing and location (cellular versus CM) of the sample collection. For most cytokines, cellular production preceded increases in the CM suggesting that cellular regulatory pathways should be studied within 6 h of stimulation. The divergent polarization-dependent expression of Vegfr1 may be essential to controlling VEGF potentially regulating angiogenesis and inflammatory cell infiltration in the vascular niche. The current study expands the repertoire of cytokines produced by polarized macrophages and provides insights into the dynamic regulation of macrophage polarization and resulting cytokines, proteins and gene expression that influence vascular inflammation.

Keywords: Chemokine; cytokine; macrophage; polarization; temporal.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Differentiation / drug effects*
  • Chemokine CCL2 / genetics
  • Chemokine CCL2 / immunology
  • Chemokine CCL4 / genetics
  • Chemokine CCL4 / immunology
  • Chemokine CCL7 / genetics
  • Chemokine CCL7 / immunology
  • Chemokine CXCL10 / genetics
  • Chemokine CXCL10 / immunology
  • Chemokine CXCL2 / genetics
  • Chemokine CXCL2 / immunology
  • Gene Expression Regulation
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics
  • Granulocyte-Macrophage Colony-Stimulating Factor / immunology
  • Humans
  • Interferon-gamma / pharmacology
  • Interleukin-10 / pharmacology
  • Interleukin-4 / pharmacology
  • Lipopolysaccharides / pharmacology
  • Macrophages / cytology
  • Macrophages / drug effects*
  • Macrophages / immunology
  • Mice
  • Phenotype*
  • Primary Cell Culture
  • Signal Transduction
  • Time Factors
  • Tissue Inhibitor of Metalloproteinase-1 / genetics
  • Tissue Inhibitor of Metalloproteinase-1 / immunology
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / immunology*
  • Vascular Endothelial Growth Factor Receptor-1 / genetics
  • Vascular Endothelial Growth Factor Receptor-1 / immunology*


  • Ccl2 protein, mouse
  • Ccl7 protein, mouse
  • Chemokine CCL2
  • Chemokine CCL4
  • Chemokine CCL7
  • Chemokine CXCL10
  • Chemokine CXCL2
  • Cxcl10 protein, mouse
  • Cxcl2 protein, mouse
  • IL10 protein, mouse
  • Lipopolysaccharides
  • Timp1 protein, mouse
  • Tissue Inhibitor of Metalloproteinase-1
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, mouse
  • Interleukin-10
  • Interleukin-4
  • Interferon-gamma
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Vascular Endothelial Growth Factor Receptor-1