IL-4 Amplifies the Pro-Inflammatory Effect of Adenosine in Human Mast Cells by Changing Expression Levels of Adenosine Receptors

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A2BsiRNA and selective A2B receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A2B and reduced expression of A2A adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine

Potentiation of degranulation by adenosine in IL-4, IgE and IL-4/IgE treated and non-treated HUCBMCs.

<p>A. IL-4/IgE treated and non-treated HUCBMCs were incubated with A23187 (60 nM) plus PMA (100 ng/ml) for 30 min. Mast cell degranulation was then evaluated by measuring hexosaminidase release. Data are from 5 different lines of cells. B. IL-4, IgE and IL-4/IgE-treated and non-treated HUCBMCs were incubated with adenosine (200 µM) for 30 min followed by the addition of A23187 (60 nM) plus PMA (100 ng/ml) to trigger mast cell degranulation. Data are from 5 different cell lines and are presented as the mean % change in hexosaminidase release over the vehicle treated cells ± SEM. ** P<0.01; * P<0.05 vs. controls, by paired student <i>t</i> test.</p

Expression levels of adenosine receptors in IL-4, IgE and IL-4/IgE treated and non-treated HUCBMCs.

<p>HUCBMCs were treated with vehicle, IL-4, IgE and IL-4/IgEfor 72 h, respectively. Total RNA was extracted from these cells and adenosine receptor expression levels were determined by real time PCR. Data are from 6–11 different lines of cells and are presented as fold change from non-treated cells ± SEM. **P<0.01 by student <i>t</i> test.</p

Adenosine potentiates anti-IgE-induced degranulation of human umbilical cord blood derived mast cells.

<p>CD133⁺ cells from human umbilical cord blood were isolated by magnetic bead separation and cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with human SCF, IL-6 and IL-3. Mast cells were identified by Kimura stain (A) and tryptase immunofluorescence (B). Cell purity at 12 weeks was >95%. All cells were then treated with human IL-4 (10 ng/ml) and IgE (5 µg/ml) for three days. Mast cell degranulation was triggered by anti-IgEmAb (1 µg/ml) and evaluated by measuring hexosaminidase release. The time course response of adenosine on anti-IgE-induced mast cell degranulation was examined by the addition of adenosine (200 µM in PBS) at the indicated time points (C). The dose response of adenosine on anti-IgE-induced mast cell degranulation was tested by adding adenosine at indicated concentration 30 min prior to anti-IgE challenge (D). Data are presented as the mean % change in hexosamindase release by adenosine over the PBS treated cells, ± SEM. *p<0.05, ** p<0.01 vs. PBS treated cells by student <i>t</i> test.</p

Effect of A₃ agonist and antagonist on anti-IgE-triggered mast cell degranulation.

<p>HUCBMCs were cultured for 12 weeks and treated with IL-4 and IgE for 3 days. A₃ selective agonist 2-Cl-IB-MECA at 3 and 10 µM was added 30 min prior to anti-IgE challenge. Mast cell degranulation was then quantitated by measuring hexosaminidase release from cells. Adenosine (10 µM) was used as a positive control. The effect of the A₃ receptor antagonist VUF5574 on adenosine-induced enhancement of anti-IgE triggered mast cell degranulation was also tested. IL-4/IgE treated HUCBMCs were incubated with VUF5574 at indicated concentrations for 30 min. The cells were then treated with adenosine (10 µM) for 30 min. Mast cell degranulation was triggered by the addition of anti-IgE. Data are from 3–6 different lines of cells and are presented as the mean % change in hexosaminidase release over the vehicle treated cells ± SEM. *p<0.05 vs. adenosine treated cells, by paired student <i>t</i> test.</p

Adenosine-induced potentiation of degranulation is abolished by A_2B antagonism.

<p>IL-4/IgE treated HUCBMCs were incubated with the A_2B receptor antagonists MRS1754 (A) and PSB1115 (B) at indicated concentration for 30 min. Cells were then treated with adenosine (10 µM) or PBS for 30 min. Mast cell degranulation was stimulated by the addition of anti-IgE. Data are from 3–4 different lines of cells and are presented as the mean % change in hexosaminidase release over the vehicle treated cells ± SEM. *p<0.05; **p<0.01 vs. PBS treated cells, by student <i>t</i> test.</p