The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages by Barbara Scheuerer, Martin Ernst, Iris Dürrbaum-Landmann, Jens Fleischer, Evelin Grage-Griebenow, Ernst Brandt, Hans-Dieter Flad, and Frank Petersen Blood Volume 95(4):1158-1166 February 15, 2000 ©2000 by American Society of Hematology

PF4-induced changes in monocyte morphology PF4-induced changes in monocyte morphology.Purified human monocytes were (A) cultured for 72 hours in the presence of 4 μmol/L PF4 or (B) left untreated. PF4-induced changes in monocyte morphology.Purified human monocytes were (A) cultured for 72 hours in the presence of 4 μmol/L PF4 or (B) left untreated. Photographs were taken by phase-contrast microscopy at the same magnification (× 20). Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology

Concentration and time-dependent effects of PF4 on the surface expression of carboxypeptidase M/MAX1 on human monocytes.(A) Concentration-kinetics. Concentration and time-dependent effects of PF4 on the surface expression of carboxypeptidase M/MAX1 on human monocytes.(A) Concentration-kinetics. Monocytes were cultured for 72 hours in the presence of increasing concentrations PF4 (•) or left untreated (-----). Cells were subsequently analyzed by flow cytometry for carboxypeptidase M/MAX1 expression, given as median fluorescence intensity as described under “Materials and Methods.” (B) Time-kinetics. Monocytes were incubated for different time periods in the absence (○) or presence (•) of a constant concentration of PF4 (4 μmol/L) and analyzed as described above. Data from 1 representative experiment out of 3 are shown. Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology

Effects of PF4 on the surface expression of different monocyte markers Effects of PF4 on the surface expression of different monocyte markers.Immunofluorescence-staining of human monocytes with antibodies directed against carboxypeptidase (A) M/MAX1, (B) HLA-DR, (C) CD14, (D) CD80, or (E) CD86 was performed either with freshly... Effects of PF4 on the surface expression of different monocyte markers.Immunofluorescence-staining of human monocytes with antibodies directed against carboxypeptidase (A) M/MAX1, (B) HLA-DR, (C) CD14, (D) CD80, or (E) CD86 was performed either with freshly isolated cells (left column) or with cells cultured for 72 hours in the absence (middle column) or presence (right column) of 4 μmol/L PF4. Cells were analyzed for the respective surface marker expression (gray histograms) or isotype controls (open histograms) by flow cytometry. Percentage values refer to the relative number of positive cells and values within brackets to mean fluorescence intensity of these cells. Data are derived from 1 representative experiment out of at least 6. Significant differences between numbers of positive cells from PF4-treated and untreated cells after culture were observed in panel A (n = 10,P < .006), panel B (n = 9, P < .008), panel C (n = 9, P < .005), and panel E (n = 11,P < .006). Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology

PF4 prevents monocytes from undergoing spontaneous apoptosis PF4 prevents monocytes from undergoing spontaneous apoptosis.Monocytes were cultured for 72 hours (A) in medium alone, (B) in the presence of 4 μmol/L PF4, or (C) in 1 ng/mL GM-CSF. PF4 prevents monocytes from undergoing spontaneous apoptosis.Monocytes were cultured for 72 hours (A) in medium alone, (B) in the presence of 4 μmol/L PF4, or (C) in 1 ng/mL GM-CSF. After simultaneous staining with annexin-V–FITC and PI, cells were analyzed by flow cytometry, as described under “Materials and Methods.” The upper right quadrant represents necrotic cells; the lower right quadrant represents apoptotic cells; and the lower left quadrant represents viable, nonapoptotic cells. Data are derived from 1 representative experiment out of 13. Statistical differences between numbers of apoptotic cells of untreated and PF4-stimulated or GM-CSF–stimulated cells were observed with P < .002 (n = 13). Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology

PF4 induces TNF-α release from monocytes PF4 induces TNF-α release from monocytes.Monocytes from 12 individual donors (indicated by different symbols) were cultured for 24 hours in the presence of 4 μmol/L PF4 or left untreated. PF4 induces TNF-α release from monocytes.Monocytes from 12 individual donors (indicated by different symbols) were cultured for 24 hours in the presence of 4 μmol/L PF4 or left untreated. TNF-α release in the supernatants was detected by ELISA. Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology

Effect of neutralizing antibodies against TNF- on PF4- and TNF--mediated rescue from monocyte apoptosis.Monocytes were cultured for 72 hours in medium alone (left panel), with 4 μmol/L PF4 (middle panel), or with 10 ng/mL TNF-α (right panel) in the presen... Effect of neutralizing antibodies against TNF- on PF4- and TNF--mediated rescue from monocyte apoptosis.Monocytes were cultured for 72 hours in medium alone (left panel), with 4 μmol/L PF4 (middle panel), or with 10 ng/mL TNF-α (right panel) in the presence (dark columns) or absence (white columns) of neutralizing antibodies directed against TNF-α. Cells were subsequently labeled with annexin-V and PI, and the percentage of apoptotic cells was determined by flow cytometry. Data from 1 representative experiment out of 6 are shown (n.d. = not determined). Barbara Scheuerer et al. Blood 2000;95:1158-1166 ©2000 by American Society of Hematology