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

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

Mário Henrique M Barros  1 , Franziska Hauck, Johannes H Dreyer, Bettina Kempkes, Gerald Niedobitek
Affiliations

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

Mário Henrique M Barros et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Examples of immunostains used to identify M1 and M2 macrophages.
Expression of CD68 or CD168 is indicated by blue cytoplasmic/membraneous staining. The expression of transcription factors pSTA1, RBP-J and CMAF is indicated by brown nuclear staining. (A) Infectious mononucleosis, a Th1 disease, reveals high numbers of CD68+pSTAT1+ macrophages as well as (B) of CD68+RBP-J+ macrophages. By contrast (C) infectious mononucleosis shows, in this field, absence of CD68+CMAF+ macrophages. Similarly, in Crohn’s disease, another Th1-associated condition (D) large numbers of CD163+pSTAT1+ macrophages as well as (E) of CD163+RBP-J+ macrophages are seen. In contrast (F) Crohn´s disease reveals, in this image, absence of CD163+CMAF+ macrophages. (G) An allergic nasal polyp, a Th2-associated disorder, shows, in this field absence of CD163+pSTAT1+ macrophages and (H) rare CD163+RBP-J+ macrophages, while (I) high numbers of CD163+CMAF+ macrophages are present. Similarly, in an allergic nasal polyp (J) in this field there is absence of CD68+pSTAT1+ macrophages and (K) only few CD68+RBP-J+ macrophages are seen. By contrast, (L) high numbers of CD68+CMAF+ macrophages are observed. (original magnification: 400x).
Figure 2
Figure 2. Dendrogram using average linkage obtained by hierarchical cluster analysis.
Two main identified clusters (I and II) are identified by brackets. Each cell population is stratified by 50th percentile, indicating high number of this cell in the tissue microenvironment of Th1 and Th2 model diseases. Num: order of variable input.
Figure 3
Figure 3. Box-plot graphs showing the numerical distribution of M1 (CD163+pSTAT1+ cells [A], CD163+RBP-J+ cells [B], CD68+pSTAT1+ cells [D] and CD68+RBP-J+ cells [E]) and M2 macrophages (CD163+CMAF+ cells [C] and CD68+CMAF+ cells [F]) according to Th model diaseases.
The P-values are from Mann-Whitney tests.
Figure 4
Figure 4. Scatter plots showing the correlation between the numbers of CD163+ and CD68+ macrophages (A), CD163+pSTAT1+ and CD68+pSTAT1+ macrophages (B), CD163+RBP-J+ and CD68+RBP-J macrophages (C), and CD163+CMAF+ and CD68+CMAF+ macrophages (D).
R2 is the constant correlation.
See this image and copyright information in PMC

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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.