Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages

PLoS One. 2013 Nov 15;8(11):e80908. doi: 10.1371/journal.pone.0080908. eCollection 2013.

Abstract

Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD / genetics
  • Antigens, CD / immunology
  • Antigens, Differentiation, Myelomonocytic / genetics
  • Antigens, Differentiation, Myelomonocytic / immunology
  • Biomarkers / metabolism
  • Cluster Analysis
  • Crohn Disease / immunology
  • Crohn Disease / pathology*
  • Gene Expression
  • Granuloma, Foreign-Body / immunology
  • Granuloma, Foreign-Body / pathology*
  • Humans
  • Hypersensitivity / immunology
  • Hypersensitivity / pathology*
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein / genetics
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein / immunology
  • Immunohistochemistry
  • Immunophenotyping
  • Infectious Mononucleosis / immunology
  • Infectious Mononucleosis / pathology*
  • Macrophages / classification
  • Macrophages / immunology
  • Macrophages / pathology*
  • Nasal Polyps / immunology
  • Nasal Polyps / pathology*
  • Oxyuriasis / immunology
  • Oxyuriasis / pathology*
  • Proto-Oncogene Proteins c-maf / genetics
  • Proto-Oncogene Proteins c-maf / immunology
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / immunology
  • STAT1 Transcription Factor / genetics
  • STAT1 Transcription Factor / immunology
  • Wound Healing / immunology

Substances

  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • Biomarkers
  • CD163 antigen
  • CD68 antigen, human
  • Immunoglobulin J Recombination Signal Sequence-Binding Protein
  • MAF protein, human
  • Proto-Oncogene Proteins c-maf
  • RBPJ protein, human
  • Receptors, Cell Surface
  • STAT1 Transcription Factor
  • STAT1 protein, human

Grant support

This work was supported by a grant from the Wilhelm Sander Foundation (2012.029.1). MB was supported by the Alexander von Humboldt Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.