AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders

5-Hydroxytryptamine Modulates Migration, Cytokine and Chemokine Release and T-Cell Priming Capacity of Dendritic Cells In Vitro and In Vivo

Beside its well described role in the central and peripheral nervous system 5-hydroxytryptamine (5-HT), commonly known as serotonin, is also a potent immuno-modulator. Serotoninergic receptors (5-HTR) are expressed by a broad range of inflammatory cell types, including dendritic cells (DCs). In this study, we aimed to further characterize the immuno-biological properties of serotoninergic receptors on human monocyte-derived DCs. 5-HT was able to induce oriented migration in immature but not in LPS-matured DCs via activation of 5-HTR1 and 5-HTR2 receptor subtypes. Accordingly, 5-HT also increased migration of pulmonary DCs to draining lymph nodes in vivo. By binding to 5-HTR3, 5-HTR4 and 5-HTR7 receptors, 5-HT up-regulated production of the pro-inflammatory cytokine IL-6. Additionally, 5-HT influenced chemokine release by human monocyte-derived DCs: production of the potent Th1 chemoattractant IP-10/CXCL10 was inhibited in mature DCs, whereas CCL22/MDC secretion was up-regulated in both immature and mature DCs. Furthermore, DCs matured in the presence of 5-HT switched to a high IL-10 and low IL-12p70 secreting phenotype. Consistently, 5-HT favoured the outcome of a Th2 immune response both in vitro and in vivo. In summary, our study shows that 5-HT is a potent regulator of human dendritic cell function, and that targeting serotoninergic receptors might be a promising approach for the treatment of inflammatory disorders

AMP influences T-cell priming capacity of human monocyte-derived dendritic cells.

<p>Immature DCs were left untreated or stimulated with 10⁻⁴ M AMP or induced to undergo maturation with LPS in the absence or the presence of AMP for 24 hours. DCs were then used to prime purified allogeneic CD4⁺CD45RA⁺ naive T-lymphocytes. After 5 days, T cells were restimulated with PMA and ionomycin, supernatants were taken and analyzed for content of IFN-γ (A), IL-5 (B) and IL-13 (C). Data are means ± SEM of triplicate culture. One representative experiment of 3 similar is shown (n = 3). For the average of all 3 experiments see the Supplemental Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037560#pone.0037560.s002" target="_blank">Fig. S2</a>).</p

Effect of AMP on CD73+/+ and CD73−/− BMDCs.

<p>A) AMP induced migration of immature BMDC generated from CD73+/+ and CD73−/− animals. DCs were stimulated with the indicated concentrations of AMP for 90 min. Results are shown as chemotatic index, calculated as the number of cells in the lower chamber containing the different stimuli divided by the number of cells in the chamber containing medium alone. One representative data out of 3 experiments is shown. B) CD73+/+ and CD73−/− BMDCs were incubated with AMP (10⁻⁴ M) or vehicle overnight. Supernatants were collected and IL-10, IL-12, TNF-α concentrations were measured in supernatants by ELISA. Data are means ± SEM of triplicate culture. One representative out of 3 experiments is shown. C) T-cell priming. OVA-DC generated from CD73+/+ and CD73−/− animals were stimulated with AMP (10⁻⁴ M) or vehicle prior to co-culture with OT-II naive T-cells for 5 days in vitro. Levels of IFN-γ, IL-5 and IL-13 were measured in the supernatants. Data are means ± SEM, n = 3. * p<0.05.</p

Contaminating adenosine is not involved in AMP induced cell function.

<p>A) Adenosine-independent migration of immature BMDC generated CD73+/+ and CD73−/− in response to AMP. DCs were stimulated with AMP (10⁻⁵ M) or Adenosine (10⁻⁶ M) in the absence or present of ADA (1 IU/ml) for 90 min. Results are shown as chemotatic index, calculated as the number of cells in the lower chamber containing the different stimuli divided by the number of cells in the chamber containing medium alone. One representative out of 3 experiments is shown (n = 3). B) CD73+/+ and CD73−/− BMDCs were incubated with OVA, adenosine (10⁻⁴ M) and AMP in the presence or absence of adenosine deaminase/ADA (1 IU/ml) overnight. Supernatants were collected and IL-10, IL-12, TNF-α concentrations were measured in supernatants by ELISA. Data are means ± SEM of triplicate culture. One representative out of 3 experiments is shown (n = 3).</p