Standardized flow cytometry assay for identification of human monocytic heterogeneity and LRP1 expression in monocyte subpopulations: Decreased expression of this receptor in nonclassical monocytes

Abstract

In this article, we present a flow cytometry assay by which human blood monocyte subpopulations-classical (CD14 11 CD16 2 ), intermediate (CD14 11 CD16 1 ), and nonclassical (CD14 1 CD16 11 ) monocytes-can be determined. Monocytic cells were selected from CD45 1 leukocyte subsets by differential staining of the low-density lipoprotein receptor-related protein 1 (LRP1), which allows reducing the spill-over of natural killer cells and granulocytes into the CD16 1 monocyte gate. Percentages of monocyte subpopulations established by this procedure were significantly comparable with those obtained by a well-standardized flow cytometry assay based on the HLA-DR monocyte-gating strategy. We also demonstrated that LRP1 is differentially expressed at cell surface of monocyte subpopulations, being significantly lower in nonclassical monocytes than in classical and intermediate monocytes. Cell surface expression of LRP1 accounts for only 20% of the total cellular content in each monocyte subpopulation. Finally, we established the within-individual biological variation (bCV%) of circulating monocyte subpopulations in healthy donors, obtaining values of 21%, 20%, and 17% for nonclassical, intermediate, and classical monocytes, respectively. Similar values of bCV% for LRP1 measured in each monocyte subpopulation were also obtained, suggesting that its variability is mainly influenced by the intrinsic biological variation of circulating monocytes. Thus, we conclude that LRP1 can be used as a third pan-monocytic marker together with CD14 and CD16 to properly identify monocyte subpopulations. The combined determination of monocyte subpopulations and LRP1 monocytic expression may be relevant for clinical studies of inflammatory processes, with special interest in atherosclerosis and cardiovascular disease. V C 2014 International Society for Advancement of Cytometry

Inflammation Intensity–Dependent Expression of Osteoinductive Wnt Proteins Is Critical for Ectopic New Bone Formation in Ankylosing Spondylitis

“…To determine whether Wnt expression was induced by inflammatory stimulation, we stimulated a monocyte cell line, RAW 264.7, and human monocytes with TNF. Human monocytes were identified using previously described methods (Supplementary Figure 2 and Supplementary Methods, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40468/abstract). After conducting a cytotoxicity assay (Supplementary Figure 3, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40468/abstract), we defined doses of 1 ng/ml and 10 ng/ml TNF as low‐intensity inflammatory stimulation and a dose of 50 ng/ml TNF as high‐intensity inflammatory stimulation.…”

Pulmonary sarcoidosis is associated with high-level inducible co-stimulator (ICOS) expression on lung regulatory T cells – possible implications for the ICOS/ICOS-ligand axis in disease course and resolution

“…As a possible limitation regarding the monocyte analysis, it should be mentioned that although the great majority of natural killer (NK) cells are confined to the lymphocyte population (based on forward-and sidescatter properties in flow cytometric analysis) we cannot exclude the possibility that some CD16 1 NK cells may be present in the monocyte gate. To avoid this potential error source, future studies should include a pan-monocyte marker, as was suggested recently [54].…”

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Immunomodulatory effects of therapeutic plasma exchange on monocytes in antiphospholipid syndrome

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