title: Temporal phenotypic features distinguish polarized macrophages in vitro.
abstract: 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.
authors.affiliation: Department of Surgery and.
authors.forename: David W
authors.initials: DW
authors.lastname: Melton
authors.forename: Linda M
authors.initials: LM
authors.lastname: McManus
authors.forename: Jonathan A L
authors.initials: JA
authors.lastname: Gelfond
authors.forename: Paula K
authors.initials: PK
authors.lastname: Shireman
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D018932:Chemokine CCL2 / Q000235:genetics / Q000276:immunology; D054407:Chemokine CCL4 / Q000235:genetics / Q000276:immunology; D054410:Chemokine CCL7 / Q000235:genetics / Q000276:immunology; D054357:Chemokine CXCL10 / Q000235:genetics / Q000276:immunology; D054426:Chemokine CXCL2 / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D010641:Phenotype; D061251:Primary Cell Culture; D015398:Signal Transduction; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000276:immunology*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000276:immunology*
chemical_list: C491768:Ccl2 protein, mouse; C515451:Ccl7 protein, mouse; D018932:Chemokine CCL2; D054407:Chemokine CCL4; D054410:Chemokine CCL7; D054357:Chemokine CXCL10; D054426:Chemokine CXCL2; C485433:Cxcl10 protein, mouse; C404915:Cxcl2 protein, mouse; C556062:IL10 protein, mouse; D008070:Lipopolysaccharides; C560825:Timp1 protein, mouse; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D016753:Interleukin-10; D015847:Interleukin-4; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D040281:Vascular Endothelial Growth Factor Receptor-1
keywords: Chemokine; cytokine; macrophage; polarization; temporal
references.citation: J Immunol. 1990 May 1;144(9):3400-10
references.pmid: 2109777
references.citation: J Leukoc Biol. 2006 Dec;80(6):1298-307
references.pmid: 16905575
references.citation: J Microbiol Immunol Infect. 2016 Jun;49(3):344-51
references.pmid: 25132397
references.citation: J Immunol. 2003 Sep 1;171(5):2610-5
references.pmid: 12928413
references.citation: Cell Mol Immunol. 2009 Oct;6(5):343-52
references.pmid: 19887047
references.citation: J Immunol. 2001 Jun 1;166(11):6861-8
references.pmid: 11359846
references.citation: PLoS One. 2013;8(10):e78045
references.pmid: 24205083
references.citation: J Exp Med. 1996 Jan 1;183(1):311-6
references.pmid: 8551238
references.citation: F1000Prime Rep. 2014 Mar 03;6:13
references.pmid: 24669294
references.citation: Science. 1993 Apr 23;260(5107):547-9
references.pmid: 8097338
references.citation: Inflamm Res. 2012 Dec;61(12):1329-38
references.pmid: 22842767
references.citation: J Clin Invest. 2012 Mar;122(3):787-95
references.pmid: 22378047
references.citation: Cell Metab. 2007 Aug;6(2):137-43
references.pmid: 17681149
references.citation: Biochem Biophys Res Commun. 2007 Oct 26;362(3):575-81
references.pmid: 17719562
references.citation: J Immunol. 1998 Sep 15;161(6):3071-6
references.pmid: 9743373
references.citation: J Clin Invest. 2008 Nov;118(11):3522-30
references.pmid: 18982158
references.citation: Annu Rev Immunol. 2009;27:451-83
references.pmid: 19105661
references.citation: Ann N Y Acad Sci. 2002 Jun;966:290-303
references.pmid: 12114286
references.citation: J Biol Chem. 2001 Apr 13;276(15):12404-9
references.pmid: 11278300
references.citation: Eur J Immunol. 1995 Jan;25(1):64-8
references.pmid: 7531149
references.citation: Nat Immunol. 2011 Mar;12(3):231-8
references.pmid: 21240265
references.citation: Br J Haematol. 1994 Nov;88(3):515-9
references.pmid: 7529532
references.citation: Circ Res. 2003 May 30;92(10):1065-7
references.pmid: 12714564
references.citation: J Immunol. 2003 Oct 1;171(7):3550-9
references.pmid: 14500651
references.citation: Endocrinology. 2005 Mar;146(3):1321-7
references.pmid: 15576460
references.citation: Immunobiology. 1997 Nov;197(5):478-93
references.pmid: 9413747
references.citation: PLoS One. 2014;9(4):e94188
references.pmid: 24736635
references.citation: Autoimmun Rev. 2010 Apr;9(6):441-8
references.pmid: 20004744
references.citation: Nature. 2005 Sep 22;437(7058):497-504
references.pmid: 16177780
references.citation: Clin Immunol. 2002 Mar;102(3):302-9
references.pmid: 11890717
references.citation: Blood. 2009 Oct 8;114(15):3244-54
references.pmid: 19567879
references.citation: Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1135-44
references.pmid: 23640482
references.citation: Skelet Muscle. 2011 Jan 24;1(1):4
references.pmid: 21798082
references.citation: J Leukoc Biol. 2004 Mar;75(3):443-52
references.pmid: 14673016
references.citation: J Exp Med. 1997 Jan 6;185(1):99-109
references.pmid: 8996246
references.citation: Biochem Biophys Res Commun. 2006 Apr 21;342(4):1137-46
references.pmid: 16516165
references.citation: J Immunol. 2008 Mar 15;180(6):4308-15
references.pmid: 18322244
references.citation: Int J Oncol. 2014 Feb;44(2):385-92
references.pmid: 24296981
references.citation: J Clin Invest. 1987 Feb;79(2):319-26
references.pmid: 3543052
references.citation: Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4560-5
references.pmid: 15070757
references.citation: BMC Genomics. 2012;13:220
references.pmid: 22672192
references.citation: Infect Immun. 2013 Jan;81(1):133-42
references.pmid: 23090958
references.citation: J Exp Med. 2009 Apr 13;206(4):937-52
references.pmid: 19349464
references.citation: PLoS Pathog. 2009 Apr;5(4):e1000393
references.pmid: 19381262
references.citation: Cell Immunol. 2012 Mar-Apr;276(1-2):101-9
references.pmid: 22608126
references.citation: Circ Res. 2004 Oct 29;95(9):858-66
references.pmid: 15514167
references.citation: Immunity. 2010 May 28;32(5):593-604
references.pmid: 20510870
references.citation: J Biol Chem. 2013 Dec 6;288(49):35428-36
references.pmid: 24151079
references.citation: Int J Mol Med. 2012 Jan;29(1):25-31
references.pmid: 21956667
references.citation: J Exp Med. 1983 Sep 1;158(3):670-89
references.pmid: 6411853
references.citation: Cancer Res. 2009 Mar 1;69(5):2133-40
references.pmid: 19223554
references.citation: Science. 1988 Sep 30;241(4874):1820-3
references.pmid: 3051378
references.citation: Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582
references.pmid: 24892271
references.citation: Nat Rev Immunol. 2013 Oct;13(10):709-21
references.pmid: 23995626
references.citation: Trends Immunol. 2002 Nov;23(11):549-55
references.pmid: 12401408
references.citation: Nat Rev Rheumatol. 2013 Dec;9(12):731-40
references.pmid: 24189842
references.citation: Immunity. 2004 Dec;21(6):831-42
references.pmid: 15589171
references.citation: Ageing Res Rev. 2009 Jan;8(1):31-5
references.pmid: 18789403
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8686-91
references.pmid: 15937121
references.citation: Clin Immunol. 2010 May;135(2):210-22
references.pmid: 20353899
references.citation: Biochem Pharmacol. 2009 Sep 15;78(6):539-52
references.pmid: 19413999
references.citation: J Pathol. 2013 Jan;229(2):176-85
references.pmid: 23096265
references.citation: Cardiol Rev. 2009 Sep-Oct;17(5):201-10
references.pmid: 19690470
references.citation: Int J Biochem Cell Biol. 2004 Oct;36(10):1882-6
references.pmid: 15203102
references.citation: J Biol Chem. 2009 Dec 4;284(49):34342-54
references.pmid: 19833726
references.citation: Blood. 2012 Sep 13;120(11):2307-16
references.pmid: 22855601
references.citation: J Immunol. 2002 Sep 1;169(5):2253-63
references.pmid: 12193690
references.citation: Blood. 2013 Dec 12;122(25):4054-67
references.pmid: 24174628
references.citation: Immunity. 2009 Aug 21;31(2):220-31
references.pmid: 19699171
references.citation: J Leukoc Biol. 2009 May;85(5):779-87
references.pmid: 19197071
references.citation: Cell Mol Life Sci. 2003 Nov;60(11):2334-46
references.pmid: 14625680
references.citation: Blood. 1996 Apr 15;87(8):3336-43
references.pmid: 8605350
references.citation: J Immunol. 2009 May 1;182(9):5393-9
references.pmid: 19380786
references.citation: Inflamm Res. 2011 Aug;60(8):769-74
references.pmid: 21487788
references.citation: Immunobiology. 2010 Sep-Oct;215(9-10):796-803
references.pmid: 20692534
references.citation: J Immunol. 2002 Apr 1;168(7):3195-204
references.pmid: 11907072
references.citation: J Surg Res. 2006 Jul;134(1):145-57
references.pmid: 16488443
references.citation: Clin Chem. 2009 Apr;55(4):611-22
references.pmid: 19246619
references.citation: Haematologica. 2010 Nov;95(11):1814-22
references.pmid: 20511666
references.citation: Immunity. 2014 Jul 17;41(1):14-20
references.pmid: 25035950
references.citation: J Immunol. 1999 Aug 1;163(3):1537-44
references.pmid: 10415057
references.citation: Cell Signal. 2012 Jun;24(6):1185-94
references.pmid: 22330073
references.citation: Nat Immunol. 2008 Aug;9(8):839-45
references.pmid: 18645592
references.citation: Nat Rev Immunol. 2010 Feb;10(2):111-22
references.pmid: 20098459
references.citation: Mol Immunol. 2013 Mar;53(3):179-86
references.pmid: 22944456
references.citation: Front Oncol. 2014 Apr 08;4:69
references.pmid: 24782982
references.citation: Vet J. 2013 Oct;198(1):245-51
references.pmid: 23985297
references.citation: Eur J Immunol. 1994 Jun;24(6):1441-5
references.pmid: 7911424
references.citation: J Exp Med. 1995 Jul 1;182(1):219-31
references.pmid: 7790818
references.citation: Respir Res. 2012;13:51
references.pmid: 22726462
references.citation: J Immunol. 2014 Apr 15;192(8):3858-67
references.pmid: 24639350
references.citation: Trends Immunol. 2014 May;35(5):190-4
references.pmid: 24703179
references.citation: J Allergy Clin Immunol. 2008 Dec;122(6):1200-1207.e1
references.pmid: 19084112
references.citation: Curr Protoc Immunol. 2008 Nov;Chapter 14:Unit 14.1
references.pmid: 19016445
references.citation: J Clin Invest. 2011 Jun;121(6):2111-7
references.pmid: 21633179
references.citation: J Leukoc Biol. 2000 Jan;67(1):53-62
references.pmid: 10647998
references.citation: J Immunol. 2007 Apr 15;178(8):5245-52
references.pmid: 17404308
references.citation: J Immunol. 2009 May 15;182(10):6237-46
references.pmid: 19414777
references.citation: Atherosclerosis. 2011 Feb;214(2):316-24
references.pmid: 21159337
references.citation: Cell Immunol. 2013 Jan;281(1):51-61
references.pmid: 23454681
references.citation: PLoS Genet. 2014 Jun;10(6):e1004368
references.pmid: 24945926
references.citation: J Autoimmun. 2009 Nov-Dec;33(3-4):222-30
references.pmid: 19819674
references.citation: Blood. 2011 May 12;117(19):5092-101
references.pmid: 21389328
references.citation: Trends Immunol. 2004 Dec;25(12):677-86
references.pmid: 15530839
references.citation: Blood. 1996 May 1;87(9):3877-82
references.pmid: 8611715
references.citation: Clin Exp Immunol. 2010 Jun;160(3):369-79
references.pmid: 20059482
references.citation: J Immunol. 2009 Sep 15;183(6):3652-60
references.pmid: 19710468
references.citation: J Immunol. 2006 Nov 15;177(10):7303-11
references.pmid: 17082649
references.citation: J Leukoc Biol. 2002 Apr;71(4):597-602
references.pmid: 11927645
references.citation: Nat Rev Immunol. 2008 Dec;8(12):958-69
references.pmid: 19029990
references.citation: Front Immunol. 2014 Mar 26;5:127
references.pmid: 24723924
references.citation: J Immunol Methods. 2008 Jan 1;329(1-2):157-66
references.pmid: 17997408
references.citation: Exp Cell Res. 2006 Nov 1;312(18):3559-69
references.pmid: 16982054
references.citation: Blood. 2014 May 15;123(20):e110-22
references.pmid: 24695852
references.citation: J Exp Med. 1992 Jul 1;176(1):287-92
references.pmid: 1613462
references.citation: FEBS Lett. 1989 Jan 2;242(2):237-9
references.pmid: 2464504
references.citation: Angiogenesis. 2001;4(2):143-54
references.pmid: 11806246
references.citation: J Biol Chem. 2002 Nov 8;277(45):42821-9
references.pmid: 12215441
references.citation: Nat Rev Immunol. 2011 Nov;11(11):738-49
references.pmid: 21984069
references.citation: J Pathol. 2008 Jan;214(2):149-60
references.pmid: 18161752
references.citation: J Immunol. 1999 Aug 15;163(4):2151-9
references.pmid: 10438956
references.citation: Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):435-44
references.pmid: 14604834
references.citation: Trends Immunol. 2013 May;34(5):216-23
references.pmid: 23218730