Figure 3.
Monocytes and lymphocytes influence vascular endothelial growth factor (VEGF) signalling. (a) Both CD14‐depleted peripheral blood mononuclear cells (PBMCs) and undepleted PBMCs were stimulated with 1 µM zoledronic acid (Zol) and 100 U/ml interleukin (IL)‐2 on day 0 and the concentrations of VEGF were measured in the supernatants on day 7. Release rates were normalized to 1 × 106cells/24 h. Data from three independent experiments are depicted as mean ± standard deviation (s.d.); *P < 0.05 compared to undepleted control. (b) Effect of lymphocyte conditioned medium on the release of Soluble VEGFR (sVEGFR)‐1 by enriched monocytes. Monocytes were enriched from blood PBMCs by immunomagnetic selection for CD14 on day 0 and cultured at 1 × 106 cells/ml. Shown are the results from three different treatment regimens: (1) unstimulated monocytes, (2) monocytes stimulated with 25% lymphocyte conditioned medium (addition of 25% cell‐free medium which was harvested on day 7 of IL‐2‐stimulated, monocyte‐depleted PBMC culture and (3) monocytes stimulated with 50% lymphocyte‐conditioned medium (addition of 50% cell‐free medium which was harvested on day 7 of IL‐2‐stimulated, monocyte‐depleted PBMC culture). Concentrations of VEGF were measured in the supernatants at the end of the respective period, the concentration of soluble VEGFR (sVEGFR)‐1 in the added medium was subtracted from this value and results normalized with regard to cell count and culture time. Data from three independent experiments are depicted as mean ± standard deviation (s.d.). *P < 0·05 compared to unstimulated control. (c) Correlation between the sVEGFR‐1 concentration in supernatants of differently stimulated (unstimulated, IL‐2 and Zol + IL‐2) PBMCs after 7 days and the detectable free VEGF concentration by enzyme‐linked immunosorbent assay (ELISA) after spiking of 400 pg/ml recombinant human VEGF (rhVEGF) into these supernatants. The reference point at 0 pg/ml sVEGFR1 is assigned to the VEGF concentration measured in the calibrator diluent of the assay. Each point represents an individual experiment. Spearman's rank correlation coefficient is r = −0·89 and P < 0·05.