Calcium-Binding Proteins S100A8 and S100A9: Investigation of Their Immune Regulatory Effect in Myeloid Cells

Abstract

High expression levels of the calcium-binding proteins S100A8 and S100A9 in myeloid cells in kidney transplant rejections are associated with a favorable outcome. Here we investigated the myeloid cell subset expressing these molecules, and their function in inflammatory reactions. Different monocyte subsets were sorted from buffy coats of healthy donors and investigated for S100A8 and S100A9 expression. To characterize S100A9high and S100A9low subsets within the CD14+ classical monocyte subset, intracellular S100A9 staining was combined with flow cytometry (FACS) and qPCR profiling. Furthermore, S100A8 and S100A9 were overexpressed by transfection in primary monocyte-derived macrophages and the THP-1 macrophage cell line to investigate the functional relevance. Expression of S100A8 and S100A9 was primarily found in classical monocytes and to a much lower extent in intermediate and non-classical monocytes. All S100A9+ cells expressed human leukocyte antigen&mdash;antigen D related (HLA-DR) on their surface. A small population (<3%) of CD14+ CD11b+ CD33+ HLA-DR− cells, characterized as myeloid derived suppressor cells (MDSCs), also expressed S100A9 to high extent. Overexpression of S100A8 and S00A9 in macrophages led to enhanced extracellular reactive oxygen species (ROS) production, as well as elevated mRNA expression of anti-inflammatory IL-10. The results suggest that the calcium-binding proteins S100A8 and S100A9 in myeloid cells have an immune regulatory effect.

Keywords: S100A9 subsets; calcium-binding proteins; immune regulation; monocytes subsets.

Figure 1

S100A9 expression is highest in CD14+ classical monocytes. (A) Classical, intermediate, and nonclassical monocytes subsets were sorted based on CD14 and CD16 expression using FACS; (B) The relative expression of S100A9 in the classical subset was 20-fold higher than that in the non-classical subset; (C) The representative FACS histogram plot showed that S100A9 expression in the three monocyte subsets overlapped with each other; (D) The median fluorescence intensity (MFI) of S100A9 in the classical subset was approximately twice as high than that in the non-classical subset; (E) The cytospin results showed that the fluorescence intensity varied greatly between individual cells within the CD14+ monocyte population; scale bar: 50 µm. The differences were tested by one-way ANOVA with Tukey’s multiple comparison tests. Data are expressed as means ± SD of at least three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

S100A9-positive monocytes express HLA-DR. Peripheral blood mononuclear cells (PBMC) were stained for CD14, HLA-DR, and S100A9, and were gated on CD14+ cell populations. (A) Representative FACS plot showing that CD14+ monocytes express both HLA-DR and S100A9. Gates were set to the upper and lower 10% of HLA-DR expression for the CD14+ monocytes, to further study subsets; (B,C) S100A9 expression was not significant between HLA-DR-high and HLA-DR-low subsets, although, S100A9 was slightly higher in the latter subset. Significant differences were calculated by t-test; (D) Representative FACS plot showing expression of CD14 and HLA-DR in gated CD33+ myeloid cells; (E) Monocytic MDSC (CD14+ HLA-DR−) subsets showed slightly higher expression of S100A9 than CD14+ HLA-DR+ subsets, but this was not significant. The differences were tested by one-way ANOVA with Tukey’s multiple comparison tests. All data are expressed as means ± SD of four different experiments. * p < 0.05, ** p < 0.01.

Figure 3

S100A9high and S100A9low subsets of classical monocytes do not differ in their expression of immune-related cytokines. (A) The addition of increased RNAse inhibitor concentration (5%) to lysis buffer minimizes the extent of RNA degradation (delta Cq < 34). Differences were tested by one-way ANOVA with Tukey’s multiple comparison tests; (B) Monocytes were sorted into two subsets based on the S100A9 protein level: S100A9high and S100A9low; (C) Relative gene expression of S100A9 from FACS sorted cell populations based on S100A9 protein levels. The S100A9high subset expressed significantly higher S100A9 mRNA levels than the S100A9low subset. The difference was tested by t-test; (D) The relative mRNA expression ratio of pro- and anti-inflammatory cytokines was not significantly different between S100A9 high and S100A9 low subsets. The dashed line indicates relative mRNA expression ratio between S100A9 high and S100A9 low subsets is one. The differences were tested using one sample t-test. All data are expressed as means ± SD of three different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Overexpression of S100A8/A9 in monocyte-derived macrophage increases reactive oxygen species (ROS) production. (A) The mRNA level in monocyte-derived macrophages after transfection was significantly higher than that in the empty plasmid control. The differences were tested by t-tests; (B) After phorbol 12-myristate 13-acetate (PMA) activation, the transfected cells were placed immediately in a luminometer to measure their ROS production real-time every 2.5 s. Data are expressed as means ± SEM of at least four different experiments; (C) The peak of ROS production, assessed at the tenth timepoint (25 s) was significantly higher in the S100A8/A9-overexpressed macrophages compared to cells transfected with the empty plasmid. Inhibition of NADPH oxidase activity by diphenylene iodonium (DPI) blocked the ROS production. The differences were tested by one-way ANOVA with Tukey’s multiple comparison tests. All data are expressed as means ± SD of at four different experiments. * p < 0.05, **** p < 0.0001.

Figure 5

Overexpression of S100A8/A9 in macrophages leads to increased IL-10 expression. (A–C) Validation of transfection efficiency of the THP-1 macrophage cell line. The following cytospins were inspected by immunofluorescence for green light emission: (A) non-transfected cells; (B) cells transfected with an empty plasmid; (C) and those with a GFP-containing plasmid. Scale bar: 50 µm. The results show high transfection efficiency in the cells; (D) Overexpression of S100A8 and S100A9 leads to a consistent increase of IL-10 expression, but not of TNFα and IL-1β. This was observed in both unstimulated cells (left panel) and LPS–stimulated cells (right panel). Data are shown as boxplots (median, upper value, lower value) representing three different experiments.