Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide, but not by proinflammatory cytokines

Abstract

Purpose: Dendritic cells (DCs) play an important role in the host's immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host.

Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor alpha, prostaglandin E2, IL-6, and IL-1beta.

Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=-0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen.

Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.

Fig. 1

Correlation between DC density and VEGF expression. The density of DCs and the quantity of VEGF expression were immunohistochemically evaluated. A significant inverse correlation was found between the density of DCs and the quantity of VEGF production analyzed by Spearman correlation coefficient (r=−0.39, p<0.05)

Fig. 2A,B

IL-12 (p70 heterodimer) production in the cultured supernatant of DCs. Maturation of monocyte-derived DCs was induced by either LPS or proinflammatory cytokine cocktail in the presence or absence of VEGF. The contents of IL-12 (p70 heterodimer) in the cultured supernatant of DCs were determined by ELISA and expressed as pg/ml. The ability of DCs cultured with LPS to produce IL-12 was inhibited by the addition of VEGF ligand in a dose-dependent manner (5 ng/ml, 10 ng/ml, and 20 ng/ml; B). In contrast, the effect of the addition of VEGF was not significantly seen in the IL-12 production from DCs matured by proinflammatory cytokine cocktails (A)

Fig. 3A,B

Allogenic T-cell response by DCs. Maturation of monocyte-derived DCs was induced by either LPS or cytokine cocktail in the presence or absence of VEGF. Allogenic T cells (1×10⁵) were coincubated with irradiated (20 Gy) DCs (2×10³) in 96-well U-bottom plates, and thymidine incorporation was expressed in cpm. The ability of DCs matured by LPS to stimulate allo-T cells was inhibited by the addition of VEGF ligand in a dose-dependent manner (5 ng/ml, 10 ng/ml, and 20 ng/ml; B). However, the ability of DCs matured by proinflammatory cytokine cocktail was not suppressed by the addition of VEGF (A)

Fig. 4A,B

Allogenic T-cell response by DCs at various ratios. Maturation of monocyte-derived DCs was induced by either LPS or cytokine cocktail in the presence or absence of VEGF (20 ng/ml). Irradiated (20 Gy) DCs were added at various ratios to allogenic T cells (1×10⁵) in 96-well U-bottom plates, and thymidine incorporation was expressed in cpm. The ability of DCs matured by LPS to stimulate allo-T cells was inhibited by the addition of VEGF (A), while the ability of DCs matured by proinflammatory cytokine cocktail was not suppressed by the addition of VEGF (B)

Fig. 5A,B

Phenotypical analysis of DCs matured by LPS. Maturation of monocyte-derived DCs was induced by LPS in the presence or absence of VEGF. Representative flow cytometric data are shown in the expression of costimulatory molecules (CD86 and CD80; A) and other molecules (CD83, HLA-DR, and MHC class I; B) on DCs. Less expression of costimulatory molecules was seen in DCs treated with LPS plus the exogenous VEGF ligand, but the expression of phenotypical antigens, HLA-DR, MHC class I, and CD83, on DCs treated with LPS plus VEGF ligand was unchanged. Representative data from three independent experiments are shown. DCs untreated with LPS (bold line), DCs treated with LPS (thin line), DCs treated with LPS in the presence of VEGF (dotted line). MFI mean fluorescence intensity

Fig. 6

Phenotypical analysis of DCs matured by proinflammatory cytokine cocktail. Maturation of monocyte-derived DCs was induced by cytokine cocktail in the presence or absence of VEGF. Representative flow cytometric data are shown in the expression of phenotypical antigens (CD86, CD80, and CD83) on DCs from three independent experiments. DCs untreated with proinflammatory cytokine cocktail (thin line), DCs treated with proinflammatory cytokine cocktail (bold line), DCs treated with proinflammatory cytokine cocktail in the presence of VEGF (dotted line)

Fig. 7A–D

Spontaneous DC apoptosis affected by the addition of VEGF. Apoptosis in DCs was measured by flow cytometry staining with annexin V and PI. The population in the right half (late and early apoptosis), of DCs treated with LPS in the presence of VEGF (20 ng/ml) was obviously greater than that of DCs treated with LPS in the absence of VEGF (A and B). The population in the right half, of DCs cultured with the cytokine cocktail with or without VEGF ligand was almost the same proportion (C and D). Representative data from three independent experiments are shown

Fig. 8

Flk-1 expression of DCs. The expression of VEGF receptor (Flk-1) on matured DCs was down-regulated in comparison to that on immature DCs