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

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

AMP regulates cytokine secretion of human monocyte-derived dendritic cells.

<p>0.2×10⁶ cells were stimulated with the indicated concentrations of AMP. LPS (3 µg/ml) or vehicle was added one hour later. Cells were incubated for 24 h and contents of TNF-α (A), IL-12p70 (B), and IL-10 (C) by unpulsed immature dendritic cells (iDCs) or LPS-pulsed mature dendritic cells (mDCs) were determined by ELISA. Data are means ± SEM of triplicate culture. One representative experiment of at least 5 similar is shown (n = 5). 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.s001" target="_blank">Fig. S1A</a>–C).</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

Characterization of the involved receptors in immature and LPS-differentiated dendritic cells by using selective antagonists.

<p>Cells were pre-incubated for 30 min with the A₁ receptor antagonist DPCPX, the A₂ receptor antagonist ZM241385, and the A₃ antagonist MRS1220 at a concentration of 10⁻⁶ M. Ca²⁺ transients were analyzed, and the ratio after stimulation with AMP at a concentration of 10⁻⁴ M for 10 s is given. Data are means ± SE (n = 3). Actin polymerization after stimulation with AMP for 25 s was measured and calculated as the ratio between the medium control and stimulated cells. Data are means ± SE (n = 3). The chemotactic index after stimulation with AMP at 10⁻⁵ M was calculated as the ratio between stimulated cells and cells incubated with medium. Data are means ± SE (n = 3). TNF-α, IL-12p70, and IL-10 release after costimulation with LPS (3 µg/ml) and 10⁻⁴ M AMP was calculated, and cytokine content is given as pg/ml/0.2×10⁶ cells. Data are means ± SEM (n = 3).</p>#<p>p<0.05 compared to control,</p>*<p>p<0.05 compared to AMP-stimulated cells.</p

AMP triggers intracellular Ca²⁺ transients in human immature monocyte-derived dendritic cells.

<p>Cells were loaded with the Ca²⁺ indicator fura-2/AM and stimulated with the indicated concentrations of AMP. One representative experiment of at least 5 similar is shown.</p

5-HT inhibits the release of IP 10/CXCL10 and stimulates MDC/CCL22 secretion in DCs.

<p>Immature and mature DCs were left untreated or were stimulated with the indicated concentration of 5-HT for 24 h (A–B). CXCL10 and CCL22 release was measured by ELISA. The results are expressed as mean pg/ml±SEM (n = 4).</p

Stimulation of serotoninergic receptors induces secretion of IL-6 in mature DCs.

<p>Mature DCs were stimulated with the indicated concentrations of 5-HT or isotype receptor agonists. Supernatants were collected 24 h after stimulation and IL-6 concentration was measured by ELISA. Results are given as mean ± SEM (n  =  4).</p

5-HT modulates IL-10 production of human monocyte-derived DCs.

<p>(A) Immature and mature DC were stimulated with the indicated concentration of 5-HT concentrations. Supernatants were collected 24 h after stimulation and IL-10 content measured by ELISA. Results are given as mean±SEM (n = 4). (B) Mature DCs were preincubated with 10⁻⁷ M of the selective5-HTR₄ antagonist RS39604 or the 5-HTR₇ antagonist SB269970 30 min prior to stimulation with 5-HT. 24 hours later production of IL-10 was measured by ELISA. Data are means±SE (<i>n</i> = 5) *p<0.05, **p<0.01, ***p<0.001.</p

5-HT modulates T-cell priming capacity of human monocyte-derived DCs.

<p>Immature DCs were left untreated or stimulated with 5-HT, or were induced to undergo maturation with LPS in the absence or the presence of indicated concentration of 5-HT for 24 h. DCs were then used to prime purified allogeneic CD4+CD45RA+ naive T cells. After 5 days, supernatants from T cells were evaluated for the secretion of IL-5 (A), IL-13 (B) and IFN-γ (C). Results are expressed as mean pg/ml±SD (n = 3). *p<0.05 between cytokines secreted by T cells stimulated with DC treated or not with 5-HT. (D) Naive mice received 10×10⁶ DO11.10 CD4⁺ T-cells intravenously on day-2. On day 0 mice were instilled intratracheally with 10⁶ OVA-pulsed, OVA-pulsed 5-HT treated DCs, or unpulsed DCs. On day4 mediastinal lymph node cells were collected and cultured for 4 days. Four days later, supernatants were harvested and analyzed for the presence of IL-4, IL-5, IL-13, and IFN-γ using commercially available ELISAs .</p