Adrenomedullin, a neuropeptide with immunoregulatory properties induces semi-mature tolerogenic dendritic cells

Abstract

Dendritic cells (DC) play a pivotal role in tolerance. Adrenomedullin (AM), a neuropeptide with anti-apoptotic and anti-inflammatory effects, may decrease T helper type 1 effector cells and induce regulatory T (Treg) cells. The aim of this study was to evaluate AM effects on murine dendritic cell (DC) maturation and functions. Bone marrow-derived DC were produced and stimulated with CpG motifs, lipopolysaccharide or AM for 24 hr. Then, DC maturation and expression of AM and AM receptors were evaluated. Compared with lipopolysaccharide-stimulated or CpG-stimulated DC, AM-stimulated DC had lower levels of co-stimulatory molecule expression and pro-inflammatory cytokine release. The AM induced high levels of interferon-γ but not of interleukin-10. Importantly, AM inhibited lipopolysaccharide-induced maturation of DC. However, allogeneic T-cell stimulation and endocytic capacity of AM-stimulated DC were comparable to those of semi-mature and mature DC. Moreover, DC expressed AM and its receptors at a basal level, and AM receptor expression increased with DC maturation. The AM stimulation induced indoleamine 2,3-dioxygenase (IDO) expression, promoting Treg cell expansion. For the first time, we describe the DC maturation phenotype by a neuropeptide (AM). We have demonstrated that AM and its receptors are expressed in DC and that exogenous AM can modify the DC phenotype and functions and can induce a semi-mature DC phenotype with IDO expression. These results indicate close interactions among immune system regulation mechanisms and calcitonin-like peptides.

Figure 1

Surface phenotype and endocytosis assay of adrenomedullin-stimulated dendritic cells (AM-DC). Bone marrow-derived DC (BMDC) were stimulated for 24 hr with bacterial agents [DNA or lipopolysaccharide (LPS)] or peptides [AM or (22-52)AM]. CD11b⁺ CD11c⁺ BMDC were collected and analysed by FACS for expression of MHC II and co-stimulatory molecules (CD40, CD80, CD86). Immature (non-stimulated, NS), DNA-stimulated, or LPS-stimulated DC were used as controls. Data are representative of three experiments, each performed in triplicate. The percentage of positive cells (a) for each marker and mean fluorescence intensity (MFI) values (b) are presented. Significant differences were identical between percentage and MFI for all markers except for CD80; in this case, MFI values display no significant differences. (c) MFI values for endocytically active DC. The DC (6 × 10⁵), differentially treated as mentioned, were incubated with 50 μg/ml FITC-conjugated BSA (64 000 molecular weight) at 30° or 4° (control) for 30 min. The data are the means (± SEM) of two experiments, each performed in triplicate. Unpaired t-test. *P<0·05, **P < 0·005 compared with non-stimulated cells; $P < 0·05, $$P < 0·0005 when indicated.

Figure 2

Surface phenotype of dendritic cells (DC) pre-treated with adrenomedullin (AM). Bone-marrow-derived DC (BMDC) were pre-incubated with AM (10⁻⁷ m) during 1, 6 or 12 hr before treatment with lipopolysaccharide (LPS; 1 μg/ml) for 24 hr and analysed by FACS for expression of maturation marker in CD11c⁺ CD11b⁺ cells as previously described in Fig. 1. Mean fluorescence intensity (MFI) values for each marker were presented. A representative experiment among three independent experiments.

Figure 3

Expression of cytokines by adrenomedullin (AM) -stimulated dendritic cells (DC). Cytokines [tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10), interferon-γ (IFN-γ)] were detected using PCR (a) and ELISA in culture supernatants (b) of bone-marrow-derived BMDC differentiated for 6 days in the presence of murine recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF; 20 ng/ml) and stimulated on day 7 as previously described. Graphs show the means ± SEM of three independent experiments. Unpaired t test. *P < 0·05 compared with non-stimulated cells (NS). LPS, lipopolysaccharide.

Figure 4

Expression of adrenomedullin (AM), calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP-1) and RAMP-2 by adrenomedullin-stimulated dendritic cells (DC). Bone-marrow-dericed DC were stimulated as previously described for 24 hr. AM expression was assayed using quantitative PCR (a) and ELISA (b). Quantitative real-time PCR for AM receptors CLR, RAMP-1, RAMP-2 and RAMP-3 were performed to quantify mRNA expression after DC stimulation (see Materials and methods section) (c). Data are the means ± SEM of three independent experiments. Unpaired t-test. *P < 0·05,**P < 0·01 lipopolysaccharide (LPS) versus non-stimulated cells (NS) and ^#P < 0·01, ^##P < 0·01 LPS versus DNA-stimulated cells.

Figure 5

Allogeneic T-cell stimulation by adrenomedullin (AM) -stimulated dendritic cells (DC). Stimulated DC were mixed with carboxyfluorescein succinimidyl ester (CFSE) -labelled allogeneic naive CD4⁺ T cells for 72 hr with different ratios of DC (DC : T, 1 : 5, 1 : 10 and 1 : 20). Percentage of proliferating CD4⁺ T cells is represented for DC : T ratio 1 : 20 (similar profile and percentages for 1 : 5 and 1 : 10 ratios). Data are representative of three independent experiments, each realized in triplicate. LPS, lipopolysaccharide.

Figure 6

Expression of indoleamine 2,3-dioxygenase (IDO) by adrenomedullin (AM) -stimulated dendritic cells (DC). (a) Expression of IDO was quantified by real-time quantitative PCR. The results were normalized for actin expression (three independent experiments). (b) Intracellular staining of IDO was assayed by flux cytometry in stimulated CD11c⁺ bone-marrow-derived DC of a representative of four independent experiments. LPS, lipopolysaccharide; NS, non-stimulated.

Figure 7

In vitro T regulatory cell induction by adrenomedullin (AM) -stimulated dendritic cells (DC). Total CD4⁺ T cells (CD25-positive and CD25-negative) were incubated with autologous stimulated DC for 72 hr at a DC : T-cell ratio of 1 : 20. After co-culturing, cells positive for CD4, CD25 or Foxp3 were counted in each co-culture, using flow cytometry. (a) Percentage of Foxp3⁺ cells among CD4⁺ CD25⁺ cells. Representative experiment showing the differential expression of Foxp3⁺ cells when gated for CD4⁺ CD25⁺ cells (R2 region). (b) Representation of the ratio of CD4⁺ CD25⁺ Foxp3⁺ T cells : CD25⁺ activated T cells. The data are representative of three independent experiments, each performed in triplicate. *P < 0·05, **P < 0·005, and ***P < 0·0005 compared with non-stimulated cells (NS). $P < 0·05 where indicated, unpaired t-test. LPS, lipopolysaccharide.

Figure 8

AM interacts with DC via CLR/RAMP expressed by DC. CLR signalling induces marked IFN production which may induced IDO upregulation via autocrine effects. IDO-positive DC promotes Foxp3 expression inducing Treg phenotype; IDO converts Trp in Kyn and blocks T effector cells. AM, adrenomedullin; APC, antigen presentating cell; CRLR, calcitonin receptor-like receptor; CTLA-4, Cytotoxic T-Lymphocyte Antigen 4; IDO, Indoleamine2-3 dioxygenase; IFN-γ, Interferon gamma; MHCII, Major Histocompatibility Class II molecules; RAMP, receptor activity-modifying protein receptor; TCR, T cell Receptor; Teff, Effector T cell; Treg, Tregulatory lymphocyte.