Macrophages in the Adult Murine Heart Figure 4. Expression of M2 macrophage markers by cTMs. (A) Heat map of M2 related gene expression by cTMs and GFP+ cells from the spleen and brain by microarray analysis. (B) Scatter/correlation profile of M2 related genes expressed by cTMs (heart) and genes expressed in GFP+ cells from the spleen and brain. (C) Micrographs of adult Cx3cr1GFP/+ mouse heart sections stained for GFP, Mrc1, CD163 and Lyve-1. Scale bar = 30 mm. (D) Flow cytometry histogram showing expression of M2 markers Mrc1 and Ly6C/G by cTMs. Histograms are representative of at least 4 independent experiments. (E) qRT-PCR analysis of Lyve-1 expression by GFP+ cells from different tissues. Mean values determined for respective tissues are normalised to means obtained for heart. Histograms show mean 6 SEM (n = 3). doi:10.1371/journal.pone.0036814.g004 e). Lyve-1 was selected since initial microarray analyses indicated that it may be a cTM-specific marker. Indeed, we found that Lyve- 1 was most highly expressed in cTMs (heart) with much lower expression in cells from adipose tissue (fat), peritoneum (PT) and lung. cTMs and GFP+ cells from the brain express high levels of specific opsonisation components, including components of complement C1q Analysis of common elements between cTMs and brain GFP+ (microglia) identified complement components, specifically com- plement C1q and its subchains (a, b and c) as highly enriched in both cell types, with greater than 20-fold enrichment compared to splenic GFP+ cells (Fig. 5 a and Table S3). Conversely, C3 was found to be highly enriched in splenic GFP+ cells and cTMs, whereas all three cell types were found to express C1q binding protein (C1qbp). Variations in complement receptors were also determined by microarray analysis (Fig. 5 b and Table S3). Complement receptor Vsig4 was uniquely detected in cTMs and complement receptor C3ar1 was highly enriched in cTMs and microglia. Complement receptors Itgam (CD11b), Itgax (CD11c), Itgb2 (CD18) and Cr1l were enriched in all three cell types. Gene expression heterogeneity in cTMs and GFP+ cells from other tissues To investigate the heterogeneity of gene expression between GFP+ cells from different tissues (heart, kidney, lung, adipose (fat), brain, peritoneum, and spleen), qRT-PCR was conducted on a selection of genes including inflammatory and tissue remodelling genes also used for validating microarray data. Both inflammatory and anti-inflammatory cytokines, IL1b and IL10, were expressed in GFP+ cells from the heart, kidney, lung and adipose tissue (Fig. 6). Chemokines Cxcl1, Cxcl2, and IL6 were also detected in these GFP+ populations at different levels. Although macrophages are regarded as a major extrahepatic source of IGF-1, IGF-1 expression was only detected in cTMs and adipose tissue GFP+ PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36814