Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

Abstract

Introduction: Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods: Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results: In the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population.

Conclusions: This is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.

Figure 1

Immunoperoxidase staining of skin in SSc patients compared with that in healthy controls. Skin sections from (a to c) healthy controls and (d to f) systemic sclerosis (SSc) patients were stained with anti-human-CD68, CD163 and CD204 antibody, respectively. A larger number of (d) CD68⁺ cells, (e) CD163⁺ cells and (f) CD204⁺ cells in SSc skin were distributed not only in the perivascular and periappendageal regions, but also between thickened collagen fibers compared with that in healthy control skin (a, b and c, respectively). Arrows indicate positively stained cells (brown); nuclei are counterstained with hematoxylin. Results are representative of 6 controls and 10 SSc patients. Bar = 100 μm.

Figure 2

Increased number of CD14⁺ cells in (PBMCs) from SSc patients. Peripheral blood mononuclear cells (PBMCs) isolated from healthy controls or systemic sclerosis (SSc) patients were analyzed by single-color flow cytometry for CD14 expression. Upper panel (a) shows the results of PBMCs from healthy controls (left), limited cutaneous systemic sclerosis (lcSSc) patients (middle), and diffuse cutaneous systemic sclerosis (dcSSc) patients (right). Values are the percentage of total PBMCs in each region. FL4, phycoerythrin-cyanin 5.1 fluorescence; SS, side scatter; #Cells = actual number of the cells. Data presented here are representative of 16 healthy controls and 41 SSc patients. Lower panel (b) depicts the summary of results, comparing percentages of CD14 positive cells in PBMCs (shown on the ordinate) from healthy controls and SSc patients. *P < 0.05 as compared with the value in cells from healthy controls.

Figure 3

Expression level of CD163 and CD204 in CD14⁺ PBMCs. (a) Left panels (healthy controls) and middle panels (systemic sclerosis (SSc) patients) depict staining with each isotype-matched monoclonal antibody control (Iso) (unfilled graph) and antigen-specific monoclonal antibody indicated (Test) (filled graph). Right panels depict staining with each monoclonal antibody in healthy controls (Test C) (fine line) versus SSc patients (Test SSc) (bold line). Data presented here are representative of 16 healthy controls and 15 (for CD163) or 41 (for CD204) SSc patients. (b) Comparison of expression level of CD163 and CD204 in CD14⁺ peripheral blood mononuclear cells (PBMCs) between healthy controls and SSc patients. Mean fluorescence intensity (MFI) are shown on the ordinate. *P < 0.05 as compared with the value in cells from healthy controls. (c) The left panel shows the representative pattern of CD204 histogram. The number of peaks was counted as 1 in the (i) left panel, (ii) 1.5 in the middle panel and (iii) 2 in the right panel. The right panel shows increased number of peaks in CD204 histogram in SSc patients. The number of peaks is shown on the ordinate. * P < 0.05 as compared with the value in cells from healthy controls.

Figure 4

Gating process for detecting CD14^brightCD163⁺CD204⁺ cells in CD14⁺hPBMCs. (a) Histogram representation of CD14⁺ cells in peripheral blood mononuclear cells (PBMCs) by three-color staining. Upper panels show the percentage of CD14⁺ cells in PBMCs from (i) healthy controls and (ii) systemic sclerosis (SSc) patients. Lower panels show enlarged histogram representation of the CD14⁺ population in PBMCs from (iii) healthy controls and (iv) SSc patients. Values over the horizontal bar are the percentage of CD14^bright cells in CD14⁺ population. (b) Dot plot representation of CD163⁺ cells in CD14⁺ population of PBMCs from healthy controls (left panel) and SSc patients (right panel). CD163⁺ cells belonged to the CD14^bright subpopulation, and the percentage of CD14^bright CD163⁺ subpopulation in SSc patients was increased compared with that in healthy controls. (c) Dot plot representation of CD163⁺ and CD204⁺ cells in CD14^brightCD163⁺ population of PBMCs from healthy controls (left panel) and SSc patients (right panel). Further extended dot plot representation of CD14^brightCD163⁺subpopulation (an upper right quadrant of Figure 4b) revealed that these cells co-express CD204. Data presented in a to c are representative of 16 healthy controls and 15 SSc patients. #Cells = actual number of the cells. (d) The graphic representation of quantitative result of CD14^brightCD163⁺CD204⁺cells in PBMCs of SSc patients and healthy controls. Percentage of CD14^brightCD163⁺CD204⁺ cells determined by flow cytometry is shown on the ordinate. P < 0.05.