SuperSAGE evidence for CD14++CD16+ monocytes as a third monocyte subset by Adam M. Zawada, Kyrill S. Rogacev, Björn Rotter, Peter Winter, Rolf-R. Marell, Danilo Fliser, and Gunnar H. Heine Blood Volume 118(12):e50-e61 September 22, 2011 ©2011 by American Society of Hematology

Purification of human monocyte subsets. Purificationof human monocyte subsets. (A) PBMCs were isolated by Ficoll-Paque and stained with anti-CD86, anti-CD14, and anti-CD16; CD86-positive cells (monocytes) are red, whereas CD86-negative (nonmonocytic) cells are black (i). NB: Percentages refer to CD86-positive monocyte subsets among all PBMCs, excluding CD86-negative cells (eg, CD16-positive NK cells and neutrophils) which protrude into the CD14+CD16++ monocyte gate in this dot plot. (B) After depletion of NK cells and neutrophils (CD16-positive nonmonocytic cells) using CD56 and CD15 MicroBeads (not shown), negatively isolated cells were separated into CD14++ (i) and CD14+/− cells (ii) using FITC-conjugated anti-CD14 Ab and accordingly anti-FITC MultiSort MicroBeads. (C) Both fractions were incubated with CD16 MicroBeads to separate CD14++ cells into CD14++CD16− (i) and CD14++CD16+ monocytes (ii), and to purify CD14+CD16++ monocytes (iii) from CD14+/− cells. Top line: Flow cytometric analysis; bottom line: microscopic images (Keyence BZ-8000J [Keyence Deutschland] equipped with a Plan Apo 60×/1.40 oil objective lens [Nikon], magnification 30×, room temperature) after cytospin and May-Grünwald-Giemsa staining. Representative examples from 12 independent experiments are shown. In each dot plot, subset-specific percentages of monocytic cells among total cells are shown as means ± SD. Adam M. Zawada et al. Blood 2011;118:e50-e61 ©2011 by American Society of Hematology

Schematic representation of differences in gene expression between the 3 monocyte subsets. Schematic representation of differences in gene expression between the 3 monocyte subsets. For each pair of monocyte subsets, the number of total transcripts, and the number of differentially expressed transcripts that reached a level of significance of P < 10−10 are depicted. Statistical analysis was performed according to Audic and Claverie.19 Adam M. Zawada et al. Blood 2011;118:e50-e61 ©2011 by American Society of Hematology

Pie charts of the functional annotation of identified transcripts from CD14++CD16+ and CD14+CD16++ monocytes based on GO categorization (biological process). Pie charts of the functional annotation of identified transcripts from CD14++CD16+ and CD14+CD16++ monocytes based on GO categorization (biological process). Using GO categories, transcripts of CD14++CD16+ and CD14+CD16++ monocytes were categorized by the function of their encoded protein products. GO terms with statistical significant difference in gene expression are highlighted and projected into the right pie chart. Fisher exact test (2-tailed test) was used to compare groups for significant enrichment of particular GO classes. Numbers of transcripts for each GO term are given. All data are presented at level 2 GO categorization. Adam M. Zawada et al. Blood 2011;118:e50-e61 ©2011 by American Society of Hematology

Monocyte subset-specific identifiers. Monocyte subset-specific identifiers. (A) Surface expression of distinct markers on CD14++CD16− monocytes (blue columns), CD14++CD16+ monocytes (red columns), and CD14+CD16++ monocytes (green columns) performed by flow cytometry. Data were measured as median fluorescence intensity (MFI) and presented as means ± SEM. Background fluorescence (measured in negative controls) was subtracted. Statistical analysis was performed using the Kruskal-Wallis test. (B) NK cells and neutrophil-depleted PBMCs before (left dot plot) and after (right dot plot) incubation with anti–HLA-DR MicroBeads and subsequent negative isolation. (C) Flow cytometric analysis of spontaneous intracellular ROS levels within the 3 monocyte subsets using the ROS-detection reagent carboxy-H2DFFDA. Data are presented and analyzed as described in panel A. (D) CD4+ T-cell proliferation, measured flow-cytometrically as cytoplasmic dilution of CFDA-SE. Monocyte subsets were isolated, stimulated with SEB (2.5 μg/mL), and cultivated with CFDA-SE–labeled CD4+ T cells for 3 days. After gating for CD3-positive cells, percentages of proliferating CD4+ T cells were determined and denoted as means ± SD. Representative examples of 5 independent experiments are shown. (E) Stimulation of isolated CD14++CD16− monocytes with 2.5 μg/mL SEB versus control. After 24, 48, and 72 hours, percentages of CD14++CD16+ monocytes (left panels) and expression of HLA-DR (right panel) of total events was determined flow-cytometrically. Percentages of CD14++CD16+ monocytes derived from CD14++CD16− monocytes are given as means ± SD. Representative examples of 5 independent experiments are shown. HLA-DR MFI was measured as described in panel A. Red arrowhead marks HLA-DR expression of unstimulated CD14++CD16+ monocytes (compare panel A). HLA-DR MFI of SEB-stimulated and control cells were compared by the paired Student t test; *P < .05, **P < .01. (F) Bottom panel: Surface expression of KDR (VEGFR2) on monocyte subsets measured by flow cytometry. Data are presented and analyzed as described in panel A. Top panel: Monocyte subsets cultivated for 3 days on Matrigel in the presence of 10 ng/mL VEGF (RPM1 medium/5% FCS). Representative examples of 3 independent experiments are shown. HUVECs were used as control cells (EGM-2 medium/5% FCS). Image acquistion was performed by the Keyence BZ-8000K microscope equipped with a Nikon Plan Apo 4×/0.2 objective and the BZ Viewer software, magnification 8-12×, room temperature. (G) Capacity to phagocyte opsonized carboxylate microspheres (0.75 μm, Yellow Green) by the 3 monocyte subsets within 30 minutes; counts of FITC-positive cells were determined flow-cytometrically and are denoted as means ± SD. Representative examples of 10 independent experiments are shown. Adam M. Zawada et al. Blood 2011;118:e50-e61 ©2011 by American Society of Hematology