In addition, predominantly immunosuppressive functions have been attributed to VEGF, such as the inhibition of dendritic cell maturation 49, induction of an immunosuppressive microenvironment and activation of regulatory T cells 40, 50. release by PBMCs First we evaluated the VEGF release kinetics of human PBMCs of healthy donors during unstimulated culture and compared it to treatment with IL\2. In addition, Zol?+?IL\2 was used for specific activation of T cells (Fig. ?(Fig.1a).1a). VEGF release was decreased between days 0 and 2 in the Zol?+?IL\2\stimulated cultures and the release of VEGF until day 7 was low in any of the different types of stimulation. Zol?+?IL\2\treated PBMCs then depicted a significant increase in VEGF release compared to the unstimulated and IL\2\stimulated cells between days 7 and 9. Following day 9, both IL\2\ and Zol?+?IL\2\stimulated cells produced more VEGF than unstimulated controls. Due to their sensitivity towards stimulation with SEP-0372814 Zol?+?IL\2, T cells might secrete higher amounts of VEGF compared ST16 to other PBMCs. To test this hypothesis, we enriched T cells from Zol?+?IL\2\treated PBMCs after 7 days. Results indicate that VEGF release from T cells is similar to unselected PBMCs treated with Zol?+?IL\2. Secretion of VEGF was lower than the release of the classical proinflammatory cytokine IFN\, which peaks around day 2 when PBMCs are stimulated with Zol?+?IL\2 (Fig. ?(Fig.11b). Open in a separate window Figure SEP-0372814 1 Vascular endothelial growth factor (VEGF) and interferon (IFN)\ production by peripheral blood mononuclear cells (PBMCs). (a) VEGF release/106 PBMCs or T cells/24 h in the indicated intervals. Concentrations of VEGF were measured in the supernatants at the end of the respective interval using enzyme\linked immunosorbent assay (ELISA) and were normalized with regard to cell count and culture time. Shown are the results SEP-0372814 from four different treatment regimens: (1) unstimulated, (2) interleukin (IL)\2 (100 U/ml IL\2 added on days 0, 7 and 9), (3) zoledronic acid (Zol)?+?IL\2 (1 M Zol added on day 0 and IL\2 100 U/ml added on days 0, 7 and 9) and (4) Zol?+?IL\2\stimulated T cells (immune\magnetically selected on day 7 from previously Zol?+?IL\2 stimulated PBMCs with addition of IL\2 100 U/ml at days 7 and 9). (b) IFN\ release/106 PBMCs/24 h in the indicated interval. Concentrations of IFN\ were measured in the supernatants at the end of the respective period and normalized with regard to cell count and culture time. Shown are the results from three different treatment regimens: (1) unstimulated, (2) IL\2 (100 U/ml IL\2 added on days 0, 7 and 9, (3) Zol?+?IL\2 (1 M Zol added on day 0 and IL\2 100 U/ml added on days 0, 7 and 9). (c) Flow cytometry analysis of either intracytoplasmic isotype or VEGF staining results from Zol?+?IL\2\stimulated PBMC measured on day 7 and SEP-0372814 gated on T cells. T cell receptor (TCR)\, cytoplasmic Isotype (cyIsotype), cytoplasmic VEGF (cyVEGF). (d) Kinetic of intracytoplasmic VEGF content for three different treatment regimens (from left to right: unstimulated, IL\2 stimulated and Zol?+?IL\2\stimulated PBMCs), depicted as mean \mean fluorescence intensity (MFI) [MFI(VEGF) C MFI(isotype control] in the respective populations ( T cells, T cells and natural killer (NK) cells) between days 1 and 14 (d1?C?d14). (e) Flow cytometry analysis showing \MFI of intracytoplasmic VEGF content of freshly isolated PBMCs. \MFI?=?MFI(VEGF)?C?MFI(isotype control). (f) Immunofluorescence analysis of the VEGF content in Zol?+?IL\2\stimulated PBMC on day 7 with anti\VEGF or isotype primary antibody and cyanin 3 (Cy3)\coupled secondary antibody. All data are presented either as a representative example of at least three independent experiments or depicted as mean??standard deviation (s.d.) of at least three independent experiments. * ?005 compared to unstimulated control, # ?005 compared to stimulation with IL\2. [Colour figure can be viewed at wileyonlinelibrary.com] Intracytoplasmic VEGF in.
In addition, predominantly immunosuppressive functions have been attributed to VEGF, such as the inhibition of dendritic cell maturation 49, induction of an immunosuppressive microenvironment and activation of regulatory T cells 40, 50