Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2010 May 1;184(9):4810-8.
doi: 10.4049/jimmunol.0901368. Epub 2010 Mar 24.

CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages

Christian A Gleissner  1 , Iftach Shaked, Kristina M Little, Klaus Ley
Affiliations
Comparative Study

CXC chemokine ligand 4 induces a unique transcriptome in monocyte-derived macrophages

Christian A Gleissner et al. J Immunol. .

Abstract

In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors, such as macrophage colony-stimulation factor (M-CSF), and chemokines, such as platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with M-CSF-induced macrophages or macrophages polarized with IFN-gamma/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for 6 d. mRNA expression was measured by Affymetrix gene chips, and differences were analyzed by local pooled error test, profile of complex functionality, and gene set enrichment analysis. Three hundred seventy-five genes were differentially expressed between M-CSF- and CXCL4-induced macrophages; 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, Ag processing and presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level; however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently upregulated or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters showed higher expression in CXCL4- than M-CSF-induced macrophages, resulting in lower low-density lipoprotein content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Primary human monocyte-derived macrophges differentiated with 100 ng/ml MCSF (M0) or 1 μM CXCL4 (M4)
(A) Morphology of macrophages after six days in culture. Bar indicates 50 μm. (B) Gene and protein expression of lineage marker genes PTPCR (CD45), CD14 (CD14), ITGAM (CD11b) in both macrophage types (differences not significant by Local Pooled Error test (LPE)). (C) The upper graph shows log2 transformed intensity of all expressed genes in M0 macrophages plotted against the intensity in M4 macrophages (r=0.934, P<0.0001). The lower plot shows the same data including only genes with a FDR<0.05 as determined by Local Pooled Error (LPE) test. (D) Heatmap showing all significantly differentially regulated genes (FDR<0.05 by LPE test). Gene expression was normalized and standardized (Gene list in Supplementary table S1). Red indicates high, green low gene expression. Genes and conditions were allowed to freely cluster in the y and x axis, respectively.
Figure 2
Figure 2. Gene ontology (GO) categories of regulated genes in M0 and M4 macrophages as determined by ProfCom analysis
Bars indicate the percentage (A, C, E) or the absolute number (B, D, F) of genes attributed to a certain GO category within all genes of the GO data set (empty bars), genes overexpressed in M0 macrophages (black bars), or genes overexpressed in M4 macrophages (grey bars). Data are arranged by biological process (A, B), cellular component (C, D), and molecular function (E, F). * P<0.05 adjusted for multiple testing.
Figure 3
Figure 3. M4 macrophages do not display a clear M1 or M2 transcriptome pattern
(A) Gene expression of selected markers for M1 and M2 polarization (TNFSF10 = TRAIL, PTX3 = pentraxin-3, MRC1 = mannose receptor). *** FDR<0.001 by LPE test. (B) Gene expression (upper row) and protein levels (bottom row) of cytokines related to M1 or M2 polarization. *** FDR<0.001 by LPE test (gene expression), * P<0.05 by paired t test (protein levels), dotted line indicates detection limit of the assay. (C) Enrichment plot of M1 or M2 gene sets in M0 versus M4 macrophages. All genes were ranked using the GSEA difference of class metric, and for each gene in the M1 or M2 gene set, the enrichment score was calculated and plotted against the position of the genes within the rank ordered data set. In both cases, no significant enrichment was found.
Figure 4
Figure 4. The M4 macrophage transcriptome is distinct from the M1 or M2 transcriptomes
(A) Modified Principal Components Analysis of M1 and M2 gene expression data (as described in Methods). Briefly, M4 gene expression data were plotted into a coordinate space defined by M1 and M2 gene expression data. (B) Hierarchical clustering of the normalized M1, M2, and M4 gene expression data. All genes were included in the analysis, and the results are displayed as dendrogram. Root = M0. #1, #2, and #3 indicate donor-specific replicates for each condition.
Figure 5
Figure 5. M0 and M4 macrophages display differential phagocytotic capacity
Macrophages differentiated with MCSF (M0) or CXCL4 (M1) were exposed to zymosan beads (A) or zymosan beads opsonized with FCS (B) as described in Materials and Methods. The phagocytotic capacity of M0 and M4 macrophages was assessed by flow cytometry. Representative histograms are shown in (A) and (B), results of two independent experiments are presented as bar graphs in (C). ** P<0.01.
Figure 6
Figure 6. Atherosclerosis-related genes in M4 macrophages and potential functional implications
(A) Expression of atherosclerosis-related genes with significantly different expression level in M0 and M4 macrophages. *FDR<0.01, ***FDR<0.001. (B) Heatmap of genes implicated in foam cell formation. Red indicates high, green low gene expression. Genes and conditions were allowed to freely cluster in the y and x axis, respectively.(C) Representative histograms of surface expression of scavenger receptor-A (SR-A) and CD36 in M0 (solid line) and M4 (dotted line) macrophages derived from the same donor. Isotype control shown in gray. (D) and (E) Mean fluorescence intensity of DiI-labeled acetylated (D) or oxidized (E) LDL in M0 (solid line) and M4 (dotted line) macrophages after 4 hours exposure to 10 μg/ml LDL as determined by flow cytometry. Representative histograms and a bar graph summarizing the flow cytometric data are shown (* P<0.05 by t test, mean ± SEM, n=3-6).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005;5:953–964. - PubMed
    1. Gerrity RG. The role of the monocyte in atherogenesis: II. Migration of foam cells from atherosclerotic lesions. Am J Pathol. 1981;103:191–200. - PMC - PubMed
    1. Gerrity RG. The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181–190. - PMC - PubMed
    1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809. - PubMed
    1. Galkina EV, Ley K. Immune and inflammatory mechanisms of atherosclerosis. Annu Rev Immunol. 2009;27:165–197. - PMC - PubMed

Publication types

MeSH terms

-