Background/Purpose:  CD163 is involved in the regulation and resolution of innate inflammation and the removal of free hemoglobin from the blood via internalization of the hemoglobin-haptoglobin complex. It is also a cell surface marker of alternatively activated macrophages overexpressed in inflammatory disorders such as systemic juvenile idiopathic arthritis. To better understand the activation of myeloid cells in rheumatic disorders, a flow cytometry panel has been developed to define polarized monocyte and macrophage phenotypes. Along with detecting surface protein markers, the novel technique called PrimeFlow™ allows specific mRNA sequences to be detected at single cell resolution. This panel was utilized to define the expression of CD163 in polarized monocyte and macrophage populations.

Methods:  A human monocytic cell line, THP-1, was cultured and stimulated in vitro with LPS and IFN-gamma, IL-4, LPS and IgG, TGFβ, or IL-10 toward M1, M2a, M2b, and M2c phenotypes, respectively. The cells were stained using the fluorochrome conjugated antibodies CD14-Pacific Blue, CD16-BV711, CD80-BV786, CD64-AF700, CD209-FITC and CD163-PE. Each condition also was stained for CD163 mRNA using the PrimeFlow™ kit’s branching technology. Cells were acquired using an analytical cytometer, and data were analyzed by FACSDiva software.

Results:  PrimeFlow™ staining of CD163 mRNA could reliably detect increased CD163 expression in THP-1 monocytic cells, primary CD14+ blood monocytes, and monocyte-derived macrophages. The PrimeFlow™ CD163 mRNA probe was also used simultaneously to detect protein surface markers, and this flow cytometry panel provided the same degree of positively stained cells as the results of staining for individual cell surface markers without mRNA labeling. This combined panel demonstrated that some alternatively activated populations, such as M2a (IL-4 conditions), show an increase in CD163 surface protein expression with no change in CD163 mRNA levels. In marked contrast and in agreement with qPCR data, increase in both CD163 mRNA and protein levels is limited to the IL-10 stimulated M2c conditions. The PrimeFlow™ technique was determined to be highly sensitive for increased mRNA expression, with a strong signal over a broad range of IL-10 concentrations in contrast to qPCR data and surface protein detection. The kinetics of CD163 induction with IL-10 stimulation were shown to be similar for both mRNA and protein. There is minimal increase in both mRNA and protein expression after 5 hours of stimulation, but significant increase after 24 hours.

Conclusion:  PrimeFlow™ is a robust and sensitive system for RNA flow cytometry, and useful for studying CD163 expression in myeloid cells. CD163 shows distinct patterns of expression in different polarized populations. At single cell resolution, CD163 protein and mRNA levels increased in IL-10 stimulated M2c cells. This flow cytometry panel consisting of CD14, CD16, CD64, CD80, CD163, and CD209 cell surface markers can be used to identify different populations of activated monocytes and macrophages in a broad range of inflammatory disorders.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-analysis-of-cd163-mrna-and-protein-expression-by-primeflow-in-polarized-monocyte-and-macrophage-populations/