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Novel macrophage polarization model: from gene expression to identification of new anti-inflammatory molecules

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Plasticity is a well-known property of macrophages that is controlled by different changes in environmental signals. Macrophage polarization is regarded as a spectrum of activation phenotypes adjusted from one activation extreme, the classic (M1), to the other, the alternative (M2) activation. Here we show, in vitro and in vivo, that both M1 and M2 macrophage phenotypes are tightly coupled to specific patterns of gene expression. Novel M2-associated markers were characterized and identified as genes controlling the extracellular metabolism of ATP to generate pyrophosphates (PPi). Stimulation of M1 macrophages with PPi dampens both NLR and TLR signaling and thus mediates cytokine production. In this context extracellular PPi enhanced the resolution phase of a murine peritonitis model via a decrease in pro-inflammatory cytokine production. Therefore, our study reveals an additional level of plasticity modulating the resolution of inflammation.

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Progressive ankylosis disease susceptibility gene product


Apoptotic speck-like protein with a caspase-activating recruiting domain


Ectonucleotide pyrophosphatase/phosphodiesterase


Lactate dehydrogenase


Classically activated macrophages


Alternatively activated macrophages


Nucleotide-binding domain and leucin-rich repeat receptors






Reactive oxygen species


Toll-like receptors


  1. Gordon S, Taylor PR (2005) Monocyte and macrophage heterogeneity. Nat Rev Immunol 5:953–964

    Article  PubMed  CAS  Google Scholar 

  2. Mantovani A, Sica A, Locati M (2007) New vistas on macrophage differentiation and activation. Eur J Immunol 37:14–16

    Article  PubMed  CAS  Google Scholar 

  3. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23–35

    Article  PubMed  CAS  Google Scholar 

  4. Martinez FO, Helming L, Gordon S (2009) Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol 27:451–483

    Article  PubMed  CAS  Google Scholar 

  5. Stout RD, Suttles J (2004) Functional plasticity of macrophages: reversible adaptation to changing microenvironments. J Leukoc Biol 76:509–513

    Article  PubMed  CAS  Google Scholar 

  6. Porcheray F, Viaud S, Rimaniol AC, Léone C, Samah B, Dereuddre-Bosquet N, Dormont D, Gras G (2005) Macrophage activation switching: an asset for the resolution of inflammation. Clin Exp Immunol 142:481–489

    PubMed  CAS  Google Scholar 

  7. Gratchev A, Kzhyshkowska J, Köthe K, Muller-Molinet I, Kannookadan S, Utikal J, Goerdt S (2006) Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals. Immunobiology 211:473–486

    Article  PubMed  CAS  Google Scholar 

  8. Pelegrin P, Surprenant A (2009) Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1beta release through pyrophosphates. EMBO J 28:2114–2127

    Article  PubMed  CAS  Google Scholar 

  9. Gilroy D, Lawrence T, Perretti M, Rossi A (2004) Inflammatory resolution: new opportunities for drug discovery. Nat Rev Drug Discov 3:401–416

    Article  PubMed  CAS  Google Scholar 

  10. Serhan CN, Brain SD, Buckley CD, Gilroy D, Haslett C, O’Neill LA, Perretti M, Rossi A, Wallace JL (2007) Resolution of inflammation: state of the art, definitions and terms. FASEB J 21:325–332

    Article  PubMed  CAS  Google Scholar 

  11. Serhan CN, Savill J (2005) Resolution of inflammation: the beginning programs the end. Nat Immunol 6:1191–1197

    Article  PubMed  CAS  Google Scholar 

  12. Martinon F, Mayor A, Tschopp J (2009) The inflammasomes: guardians of the body. Annu Rev Immunol 27:229–265

    Article  PubMed  CAS  Google Scholar 

  13. Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, Panther E, Di Virgilio F (2006) The P2X7 receptor: a key player in IL-1 processing and release. J Immunol 176:3877–3883

    PubMed  CAS  Google Scholar 

  14. Pelegrin P, Surprenant A (2009) The P2X(7) receptor-pannexin connection to dye uptake and IL-1beta release. Purinergic Signal 5:129–137

    Article  PubMed  CAS  Google Scholar 

  15. Pelegrin P, Barroso-Gutierrez C, Surprenant A (2008) P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage. J Immunol 180:7147–7157

    PubMed  CAS  Google Scholar 

  16. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  17. de Hoon MJ, Imoto S, Nolan J, Miyano S (2004) Open source clustering software. Bioinformatics 20:1453–1454

    Article  PubMed  Google Scholar 

  18. Saldanha AJ (2004) Java treeview–extensible visualization of microarray data. Bioinformatics 20:3246–3248

    Article  PubMed  CAS  Google Scholar 

  19. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:14863–14868

    Article  PubMed  CAS  Google Scholar 

  20. Kolaczkowska E, Koziol A, Plytycz B, Arnold B (2010) Inflammatory macrophages, and not only neutrophils, die by apoptosis during acute peritonitis. Immunobiology 215:492–504

    Article  PubMed  CAS  Google Scholar 

  21. Bystrom J, Evans I, Newson J, Stables M, Toor I, Van Rooijen N, Crawford M, Colville-Nash P, Farrow S, Gilroy DW (2008) Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP. Blood 112:4117–4127

    Article  PubMed  CAS  Google Scholar 

  22. Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362:299–309

    Article  PubMed  CAS  Google Scholar 

  23. Sim J, Park C, Oh S, Evans RJ, North RA (2007) P2X1 and P2X4 receptor currents in mouse macrophages. Br J Pharmacol 152:1283–1290

    Article  PubMed  CAS  Google Scholar 

  24. Ho AM, Johnson MD, Kingsley DM (2000) Role of the mouse ank gene in control of tissue calcification and arthritis. Science 289:265–270

    Article  PubMed  CAS  Google Scholar 

  25. Martinez FO, Sica A, Mantovani A, Locati M (2008) Macrophage activation and polarization. Front Biosci 13:453–461

    Article  PubMed  CAS  Google Scholar 

  26. Martinez FO, Gordon S, Locati M, Mantovani A (2006) Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. J Immunol 177:7303–7311

    PubMed  CAS  Google Scholar 

  27. Scotton CJ, Martinez FO, Smelt MJ, Sironi M, Locati M, Mantovani A, Sozzani S (2005) Transcriptional profiling reveals complex regulation of the monocyte IL-1 beta system by IL-13. J Immunol 174:834–845

    PubMed  CAS  Google Scholar 

  28. Humphreys BD, Dubyak GR (1998) Modulation of P2X7 nucleotide receptor expression by pro- and anti-inflammatory stimuli in THP-1 monocytes. J Leukoc Biol 64:265–273

    PubMed  CAS  Google Scholar 

  29. Taniguchi S, Sagara J (2007) Regulatory molecules involved in inflammasome formation with special reference to a key mediator protein, ASC. Semin Immunopathol 29:231–238

    Article  PubMed  CAS  Google Scholar 

  30. Fernandes-Alnemri T, Wu J, Yu J, Datta P, Miller B, Jankowski W, Rosenberg S, Zhang J, Alnemri E (2007) The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14:1590–1604

    Article  PubMed  CAS  Google Scholar 

  31. MacKenzie AB, Young MT, Adinolfi E, Surprenant A (2005) Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors. J Biol Chem 280:33968–33976

    Article  PubMed  CAS  Google Scholar 

  32. Humphreys BD, Dubyak GR (1996) Induction of the P2z/P2X7 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN-gamma in the human THP-1 monocytic cell line. J Immunol 157:5627–5637

    PubMed  CAS  Google Scholar 

  33. Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A (2010) P2X7 receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. J Immunol 185:2611–2619

    Article  PubMed  CAS  Google Scholar 

  34. Milner JD, Orekov T, Ward JM, Cheng L, Torres-Velez F, Junttila I, Sun G, Buller M, Morris SC, Finkelman FD, Paul WE (2010) Sustained IL-4 exposure leads to a novel pathway for hemophagocytosis, inflammation and tissue macrophage accumulation. Blood 116:2476–2483

    Article  PubMed  CAS  Google Scholar 

  35. Varin A, Mukhopadhyay S, Herbein G, Gordon S (2010) Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion. Blood 115:353–362

    Article  PubMed  CAS  Google Scholar 

  36. Gea-Sorli S, Closa D (2009) In vitro, but not in vivo, reversibility of peritoneal macrophages activation during experimental acute pancreatitis. BMC Immunol 10:42

    Article  PubMed  Google Scholar 

  37. Prosdocimo DA, Douglas DC, Romani A, O’Neill WC, Dubyak GR (2009) Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells. Am J Physiol Cell Ph 296:C828–C839

    Article  CAS  Google Scholar 

  38. Sun SC, Ley SC (2008) New insights into NF-kappaB regulation and function. Trends Immunol 29:469–478

    Article  PubMed  CAS  Google Scholar 

  39. Maksymowych W (2002) Bisphosphonates–anti-inflammatory properties. Curr Med Chem 1:15–28

    CAS  Google Scholar 

  40. Russell RG, Watts NB, Ebetino FH, Rogers MJ (2008) Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 19:733–759

    Article  PubMed  CAS  Google Scholar 

  41. Pennanen N, Lapinjoki S, Urtti A, Mönkkönen J (1995) Effect of liposomal and free bisphosphonates on the IL-1 beta, IL-6 and TNF alpha secretion from RAW 264 cells in vitro. Pharm Res 12:916–922

    Article  PubMed  CAS  Google Scholar 

  42. Serretti R, Core P, Muti S, Salaffi F (1993) Influence of dichloromethylene diphosphonate on reactive oxygen species production by human neutrophils. Rheumatol Int 13:135–138

    Article  PubMed  CAS  Google Scholar 

  43. Kachur AV, Manevich Y, Biaglow JE (1997) Effect of purine nucleoside phosphates on OH-radical generation by reaction of Fe2 + with oxygen. Free Radic Res 26:399–408

    Article  PubMed  CAS  Google Scholar 

  44. Dombrecht EJ, De Tollenaere CB, Aerts K, Cos P, Schuerwegh AJ, Bridts CH, Van Offel JF, Ebo DG, Stevens WJ, De Clerck LS (2006) Antioxidant effect of bisphosphonates and simvastatin on chondrocyte lipid peroxidation. Biochem Biophys Res Commun 348:459–464

    Article  PubMed  CAS  Google Scholar 

  45. Gloire G, Piette J (2009) Redox regulation of nuclear post-translational modifications during NF-kappaB activation. Antioxid Redox Signal 11:2209–2222

    Article  PubMed  CAS  Google Scholar 

  46. Kabe Y, Ando K, Hirao S, Yoshida M, Handa H (2005) Redox regulation of NF-kappaB activation: distinct redox regulation between the cytoplasm and the nucleus. Antioxid Redox Signal 7:395–403

    Article  PubMed  CAS  Google Scholar 

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We thank Dr. Olga Fernández and Ms. Ana I. Gomez for in vivo models, technical support, and advice. We appreciate Prof. Annmarie Surprenant and Prof. Alex Verkhratsky for their support during this work. We are indebted to Dr. David Brough for carefully revising the manuscript. This work was supported by grants from Instituto Salud Carlos III FEDER (EMER07/049 and PFIS09/00120) and Fundación Séneca (11922/PI/09), managed by Fundación Formación Investigación Sanitaria Región de Murcia (FFIS).

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The authors declare no competing financial interests.

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Correspondence to Pablo Pelegrín.

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G. Lopez-Castejón and A. Baroja-Mazo contributed equally to this work.

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Supplemental Fig. 1. Expression of different housekeeping genes during macrophage polarization gradient. A Real-time quantitative (qRT)-PCR for the indicated house-keeping genes (GAPDH, YWHAZ, HPRT1, and SDHA) during the polarization gradient protocol. B qRT-PCR for TNF-α gene expression in M1, M1/M2, or M2 polarized macrophages normalized to the four different housekeeping genes. No significant differences were found in the fold increase/decrease trend among the different macrophage phenotypes. (TIFF 1259 kb)


Supplemental Fig. 2. Expression of the 39 analyzed genes in extreme M1 or M2 macrophages. Real-time quantitative RT-PCR for the indicated genes in extreme M1 (A) or M2 (B) polarization. M2 genes are represented in green, M1 in red, and housekeeping genes in blue. Relative expression normalized to YWHAZ was log2 transformed. (TIFF 1398 kb)


Supplemental Fig. 3. Gene expression in extreme M1 or M2 macrophages. Dot plot representing log2 transformed expression data from real-time quantitative RT-PCR normalized values for all genes included in this study (Suppl. Fig. 2). Data for each individual gene are represented in accordance with its expression in extreme M1 or M2 genes. R 2 = 0.341. (TIFF 2157 kb)

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Lopez-Castejón, G., Baroja-Mazo, A. & Pelegrín, P. Novel macrophage polarization model: from gene expression to identification of new anti-inflammatory molecules. Cell. Mol. Life Sci. 68, 3095–3107 (2011).

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