The Influence Of VEGF-C/VEGFR3 Signaling On The Mononuclear Cell Derived Dendritic Cells From Cancer Patients

Objective: To testify the expression of lymphatic endothelial marker VEGFR3 on the mononuclear cell derived dendritic cells from cancer patients. Add the VEGFR3 ligand, VEGF-C, to the culture medium, and detect the influence of VEGF-C/VEGFR3 signaling on DC function. Then further detect the relationship of VEGF-C/VEGFR3 with the TLR expression level to elucidate the mechanism that leads to the DC malfunction.Methods:50 cases of the peripheral blood from cancer patients were collected, and we use rh GM-CSF and rh IL-4 to harvest dendritic cells. LPS, the specific TLR4 agonist, was used to get mature DC, he expression of VEGFR3 on DC was detected by FACS, and this also testified by the VEGFR3 immunofluorescence. Then we further test the influence of VEGF-C/VEGFR3 signaling on the DC function. We add exogenous VEGF-C into the culture medium, FACS was used to detect the influence of VEGF-C on DC phenotype; MTT was used to test the influence of VEGF-C on DC stimulated allogenic T lymphocyte proliferation ability. DC survival rate was tested by counting the number of the cells. ELISA was used to test the influence of VEGF-C on the cytokine secretion by DC. FACS was used to analyze the relationship of VEGF-C/VEGFR3 signaling with the expression level of TLR4 on DC.Results: The lymphatic endothelial marker VEGFR3 was expressed on the tumor peripheral mononuclear derived dendritic cells. The expression of VEGFR3CD11 c on immature DC is(8.52±4.04)%, after LPS stimulation, it increased to(18.56±5.25)%on mature DC(P<0.05). The immunofluorescence also showed the VEGFR3 green fluorescent protein in the cell membrane and cytoplasm, and the expression level in the LPS group is more than the control group. VEGF-C has an inhibitory effect on the DC stimulated T lymphocyte proliferation ability, the stimulation index SI in the LPS group is 3.22±0.33, and it is decreased to 1.57±0.38 after the addition of VEGF-C(P<0.05), this indicate that VEGF-C inhibit the LPS stimulated DC activity in a certain mechanism. Besides, VEGF-C has an inhibitory effect on the survival rate of DC cells.VEGF-C/VEGFR3 signaling can also decrease the secreted cytokine level of IL-6?TNF-??IL-12 of DC. There is an upregulation of cytokine level after LPS stimulation:the secreted IL-6 increased from(44.73±34.02) pg/ml to(350.32±53.84) pg/ml,TNF-? increased from(25.36±24.61) pg/ml to(613.57±101.35) pg/ml, IL-12 increased from(41.97±8.02) pg/ml to(233.97±108.36) pg/ml. But the further addition of VEGF-C into the LPS group can decrease the cytokine level, of which IL-6 decreased to(147.41±27.28) pg/ml, TNF-? decreased to(244.13±36.20) pg/ml,IL-12 decreased to(100.65±18.96) pg/ml. Moreover, VEGF-C has no effect on the expression of CD80 and CD83 on DC. In addition, VEGF-C could decrease the expression of TLR4 on DC. The expression of TLR4 in the control group is(2.50±2.61)%, after LPS stimulation, it increased to(14.10±4.75)%, which shows a statistical significance(P<0.05). But if we further added VEGF-C in the LPS group,it downregulate the expression of TLR4 to a level of(5.08±3.22)%(P<0.05), this indicate that the VEGF-C/VEGFR3 signaling may negatively regulate DC function through the downregulation of TLR4.Conlusion: The mononuclear cell derived dendritic cells from cancer patients express the lymphatic endothelial marker VEGFR3. VEGF-C/VEGFR3 can inhibit the upregulation of DC function which elicits by LPS. This may achieved by the downregualtion of TLR4 by VEGF-C, and act as a negative regulator of DC function.But the specific molecular mechanism needs further exploration.