AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells

Abstract

Background and purpose: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs).

Experimental approach: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity.

Key results: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-kappaB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-kappaB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs.

Conclusions and implications: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

Figures

Figure 1

AM3 upregulates cell surface molecules on human MDDCs. (a) MDDCs were cultured for 16 h in the presence of AM3 (1 μg mL−1, dotted line), LPS (0.1 μg mL−1, bold line) or medium alone (thin line), and surface markers were analysed by flow cytometry as stated in Materials and methods. A representative experiment from one of the eight independent donors is shown. (b) Quantification of the experiments shown in (a). The data represent the mean intensity fluorescence (MIF)±s.d. of eight independent donors. *P<0.0001 vs untreated cells (UT) MDDCs: paired t-test). LPS, lipopolysaccharide; MDDC, monocyte-derived dendritic cell.

Figure 2

AM3 diminishes FITC-dextran uptake by MDDCs. (a) MDDCs were left untreated or stimulated with AM3 (1 μg mL−1) or LPS (0.1 μg mL−1) for 16 h and FITC-dextran uptake was measured by flow cytometry. The shading represents FITC-dextran uptake at 4 °C. One representative experiment is shown. (b) Quantification of the experiments shown in (a). UT, untreated cells. The data represents the mean intensity fluorescence (MIF)±s.d. of three independent experiments. FITC, fluorescein isothiocianate; LPS, lipopolysaccharide; MDDC, monocyte-derived dendritic cell.

Figure 3

AM3 induces IL-12p70 and IL-10 production by human DCs. Human MDDCs were treated with AM3 (1 μg mL−1) or LPS (0.1 μg mL−1) for 24 h and then IL-12p70 (left) and IL-10 (right) production was analysed by ELISA. Experiments were performed in triplicate and the data represent the mean±s.d. from four independent donors. DC, dendritic cell; IL, interleukin; LPS, lipopolysaccharide; MDDC, monocyte-derived DC.

Figure 4

AM3 increases the proliferation of allogeneic T cells and IFN-γ production. (a). MDDCs were cultured in medium with or without AM3 (1 μg mL−1) or LPS (0.1 μg mL−1) for 24 h. An MLR was conducted for 5 days, as described in Materials and methods, and the background level of [3H] TdR uptake was determined by measuring the reactions without stimulation. The values are the mean of triplicate±s.d. The experiment was carried out with four independent donors and one representative experiment is shown. (b). ELISA analysis of IFN-γ in 6-day MLR supernatants (stimulator/responder ratio 1/80 and 1/20). The experiment was performed on five different donors and two representative donors are shown. The values are the mean of duplicate±s.d. IFN, interferon; LPS, lipopolysaccharide; MDDC, monocyte-derived dendritic cell; MLR, mixed lymphocyte reaction.

Figure 5

AM3 induces chemokine and chemokine receptor mRNA expression by human immature DCs. Immature MDDCs were either left untreated or they were exposed to 0.1 μg mL−1 LPS or 1 μg mL−1 AM3. After 12 h, expression of mRNA encoding for the indicated chemokines (a) or chemokine receptors (b) was evaluated in RNase protection assays. The relative levels of chemokine and chemokine receptor mRNA were assessed by scanning densitometry, and the results are expressed in arbitrary units (numbers below the tracks) as the induction over the values obtained in untreated (UT)-MDDCs. Values were normalized to the expression of the ribosomal housekeeping protein L32 in each sample. A representative experiment of three independent donors is shown. DC, dendritic cell; LPS, lipopolysaccharide; MDDC, monocyte-derived DC.

Figure 6

AM3 increases the nuclear localization of p65/reaA, and promotes IkBα degradation and p38 MAPK phosphorylation. (a) MDDCs were left untreated (UT) or stimulated with AM3 (1 μg mL−1) for 1 h, and the distribution of p65 was analysed by indirect immunofluorescence (red). The nuclei were stained with DAPI (blue) and the representative fields of one of the two independent experiments are shown. (b) MDDCs were either untreated or stimulated with LPS (0.1 μg mL−1) or AM3 (1 μg mL−1) for different times and protein extracts were analysed by western blots probed with anti-IκBα or with (c) Anti-phospho-ERK1/2 (p-ERK), anti-ERK1/2 (ERK), anti-phospho-p38 (p-p38), anti-p38 (p38) antibodies. Representative results of three independent experiments performed are shown. LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MDDC, monocyte-derived dendritic cell.

Figure 7

AM3 induces TLR-4-mediated NF-κB activation. (a) TLR-4 (upper) and TLR-2 (bottom) stably transfected HEK-293 cells were transfected with κB-Luc plasmid. These cells were exposed to AM3 (1, 10, 100 μg mL−1) and NF-κB activity was evaluated by measuring luciferase levels. The luciferase activity is represented as the induction above the values obtained in the absence of stimulation, and the values are the mean of triplicate experiments (±s.d.). As controls, cells were stimulated with TLR-4 (LPS: 10–100 ng mL−1) and TLR-2 agonists (PamCys3: 1–10 μg mL−1). A representative result from one of the three independent experiments is shown. (b) Neutralizing antibodies against TLR4 abolished the upregulation of the surface expression of CD80, CD83 and CD86 induced by AM3 in MDDCs. Cells were left treated with LPS (10 ng mL−1) or AM3 (1 μg mL−1) for 16 h and, additionally, were preincubated with neutralizing anti-TLR4 or isotyped matched antibodies (10 μg mL−1) 1 h before treatment. Cell surface expression was analysed by immunofluorescence. Data are the mean intensity fluorescence (MIF)±s.d. of four independent experiments. LPS, lipopolysaccharide; MDDC, monocyte-derived dendritic cell; TLR, Toll-like receptor.

Figure 8

AM3 upregulates cell surface markers and allogeneic T-cell proliferation in MDDCs from chronic HCV-infected patients. (a) MDDCs were cultured for 16 h in the presence of AM3 (1 μg mL−1), LPS (0.1 μg mL−1) or medium alone (UT), and the surface markers expressed were analysed by flow cytometry as stated in Materials and methods. The data represent the mean intensity fluorescence (MIF). A representative experiment from one of the eight independent donors is shown. (b). MDDCs were cultured in medium alone (UT), with LPS (0.1 μg mL−1) or AM3 (1 μg mL−1) for 24 h. An MLR was conducted for 5 days as described in Materials and methods. The background level of [3H] TdR uptake was determined by measuring the reactions without stimulation. The values are the mean of triplicate experiments (±s.d.). The experiment was carried out on five independent patients and one representative experiment is shown. HCV, hepatitis C virus; LPS, lipopolysaccharide; MLR, mixed lymphocyte reaction; MDDC, monocyte-derived dendritic cell.