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. 2012 Apr 11;14(2):R74.
doi: 10.1186/ar3796.

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Carmen A Ambarus  1 , Troy Noordenbos, Maria J H de Hair, Paul P Tak, Dominique L P Baeten
Affiliations

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Carmen A Ambarus et al. Arthritis Res Ther. .

Abstract

Introduction: Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods: Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results: The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions: The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.

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Figures

Figure 1
Figure 1
Immunohistochemical analysis of the expression of polarization markers in spondyloarthritis (SpA) compared with rheumatoid arthritis (RA) synovial tissue. The expression of CD68, CD163, and CD32 in the intimal lining layer (A through C) and the expression of CD68, CD64, CD200R, CD14, CD163 and CD32 in the synovial sublining (D through I) as assessed with semiquantitative scoring (0 to 3) of immunohistochemical stainings. Data are represented as median and interquartile range of 18 SpA and 20 RA patients.
Figure 2
Figure 2
Monocyte subsets and expression of phenotypic markers on spondyloarthritis (SpA) and rheumatoid arthritis (RA) patients and healthy donor monocytes. The percentage of CD14+CD16-, CD14+CD16+, and CD14dimCD16+ monocytes from SpA and RA patients and healthy donors was measured with flow cytometry (A). Expression of CD64, CD200R, and CD16 on monocytes from SpA and RA patients and healthy donors was measured with flow cytometry and expressed as geometric mean fluorescence intensity (gMFI). Data are represented as median and interquartile range of at least seven independent experiments (B).
Figure 3
Figure 3
Expression of phenotypic markers on in vitro polarized monocyte-derived macrophages from spondyloarthritis (SpA) and rheumatoid arthritis (RA) patients and healthy individuals. The expression of MΦIFN-γ markers CD80 and CD64, MΦIL-4 markers CD200R and CD14, and MΦIL-10 markers CD163 and CD16 was measured with flow cytometry on in vitro polarized monocyte-derived macrophages. Data are expressed as geometric mean fluorescence intensity (gMFI) of the marker expression after polarization with IFN-γ, IL-4, or IL-10, respectively, divided by the gMFI of the marker expression on unpolarized macrophages. Data are represented as median and interquartile range of at least five independent experiments.
Figure 4
Figure 4
Double immunofluorescence stainings of CD68 and macrophage polarization markers on synovial tissue from chronic inflammatory arthritis. Colocalization of CD68 (green) with MΦIFN-γ marker CD64 (A), MΦIL-4 markers CD200R (B) and CD14 (C), and MΦIL-10 markers CD163 (D), CD16 (E), and CD32 (F) (red) on synovial tissue macrophages. Figures are representative of five spondyloarthritis (SpA) and five rheumatoid arthritis (RA) patients. Higher-magnification photos are included in each figure part.
Figure 5
Figure 5
Expression of polarization markers on CD68+ cells in synovial tissue from chronic inflammatory arthritis. Figure represents the percentage of CD64+ (A), CD200R+ (B), CD14+ (C), and CD163+ (D) cells from the total number of CD68+ cells in five spondyloarthritis (SpA) and five rheumatoid arthritis (RA) patients. Data were acquired by manual quantitative scoring of double-immunofluorescence stainings and are represented as median and interquartile range.
Figure 6
Figure 6
Double-immunofluorescence stainings of MΦIFN-γ, MΦIL-4, and MΦIL-10 polarization markers on synovial tissue from chronic inflammatory arthritis. Colocalization of MΦIFN-γ marker CD64 with CD200R (A), CD163 (B), and CD32 (C), colocalization of MΦIL-4 marker CD200R with CD14 (D), CD163 (E), and CD32 (F), and colocalization of MΦIL-10 marker CD163 with CD14 (G) and CD32 (H). Figure parts are representative of five spondyloarthritis (SpA) and five rheumatoid arthritis (RA) patients.
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