Adenosine stimulates chloride conductance through the A_2B adenosine receptor (A_2BAR).

<p>The air-liquid interface cultures of Calu-3 cells were stimulated with 100 µM of apical adenosine, followed by apical addition of 50 µM of alloxazine, a specific inhibitor for A_2BAR. The adenosine-stimulated I_SC was decreased by ∼75% (n = 6).</p

Adenosine-stimulated transepithelial I_SC was mediated by CFTR channel.

<p>Calu-3 epithelia were stimulated with 100 µM of apical adenosine, resulting in a significant increase in I_SC above baseline. Such an adenosine-induced I_SC was mostly absent in CFBE41o cells in which CFTR channel was dysfunctional or was significantly inhibited with 25 µM of CFTR inhibitor CFTR_inh172. Student's t-test was performed to determine the statistic significance (<i>p</i><0.05, n = 5).</p

Effect of ethanol pre-exposure on adenosine-induced epithelial ion transport.

<p>Calu-3 cells were cultured at an air-liquid interface on membrane filters and basolaterally exposed to 0, 25, 50 and 100 mM of ethanol for 24 hours. These Calu-3 epithelia were placed in an Ussing chamber with asymmetrical buffers of chloride (apical 10.4 mM and basolateral 139.8 mM). Following voltage clamp, stable short circuit baselines were attained in 15 to 20 min. I_SC was measured after blocking Na⁺ channels with 100 µM of apical amiloride and stimulated with 100 µM of apical adenosine. Alterations in ion transport are expressed as difference in I_SC from their baseline. Ethanol pre-exposure decreased 100 µM adenosine mediated epithelial ion transport in a dose-dependent manner. Asterisks indicate significant differences between groups by One-way ANOVA test (<i>p</i><0.05, n = 5 for each condition).</p

Adenosine-stimulated short circuit current is chloride conductance.

<p><b>A</b>) I_SC of Calu-3 epithelia was measured by using the asymmetric chloride buffers. After stimulated with 100 µM of apical adenosine, the cells gave rise to I_SC of ∼75 µA/cm². The I_SC was blocked by ∼58% by basolateral addition of bumetanide (100 µM), but not by acetazolamide (20 µM) or DNDS (100 µM). <b>B</b>) I_SC of Calu-3 epithelia was measured with either asymmetric chloride buffers or symmetric chloride buffers. The adenosine-stimulated I_SC with no chloride-gradient buffers was ∼92% lower than that with chloride-gradient buffers. Significance of the difference was determined by Student's t-test (<i>p</i><0.01, n = 5).</p

Phosphodiesterase inhibitors restore the ethanol suppression of adenosine-induced transepithelial chloride conductance.

<p>The air-liquid interface cultures of Calu-3 cells were exposed to 0 and 100 mM of ethanol for 24 hours. Adenosine-stimulated I_SC was measured, showing that ethanol-exposed Calu-3 epithelia had significantly lower adenosine-stimulated I_SC than the no ethanol control. However, phosphodiesterase inhibitors, IBMX (100 µM) or papaverine (50 µM), fully restored the ethanol-suppressed adenosine-stimulated I_SC. Asterisks indicate statistically significant differences between groups (p<0.05, n = 4 for each group).</p

Effect of ethanol exposure on cellular cAMP level and adenosine analog antagonizes ethanol suppression of adenosine-induced chloride secretion.

<p><b>A</b>) Calu-3 epithelia, exposed to 0 and 100 mM of ethanol for 24 hours, were apically stimulated with 100 µM of adenosine for 10 min. Cells were lysed and cAMP levels were estimated by immunoassay. Ethanol pre-exposure significantly decreased the adenosine-induced cellular cAMP level to ∼64% of the no ethanol control (<i>p</i><0.05, n = 5). <b>B</b>) In Calu-3 epithelia, ethanol pre-exposure decreased the adenosine-stimulated I_SC. Such an inhibitory effect of ethanol on adenosine-induced I_SC was antagonized by Sp-cAMPS, a phoshodiesterase-resistant cAMP analog (<i>p</i><0.05, n = 6).</p

Impairment of phagosomal HOCl production in neutrophils from Myeloid Cftr−/− mice.

<p>Neutrophil production of HOCl was assessed using the fluorescent HOCl probe R19-S. Phagocytosis was performed by mixing neutrophils with opsonized PsA in the chloride-free buffer (0 ) for 15 min, and the cells were further incubated in the buffer containing either 0 mM or 122 for 20 min. Then, R19-S was added to incubate for an additional 15 min. Fluorescence of R19-S produced by HOCl oxidation was analyzed by flow cytometry. Mean fluorescence intensity (MFI) of each sample was obtained and compared to their 0-min time point. Statistical difference was judged by Student’s t-test. Double asterisks indicate significant differences (p<0.01, n = 4).</p

Lung host defense defect in Myeloid-Cftr−/− mice.

<p>Myeloid-Cftr−/− mice and Cftr^fl10 mice were infected with the agarose-embedded PsA at the lethal dose of (5×10⁶ cfu) by lung intubation. Casualties were recorded and survival curves traced. The log-rank test was performed to check statistical difference in survival between the Cftr^fl10 mice (n = 34) and the myeloid-Cftr−/− mice (<i>n</i> = 35). Double asterisks mean significant difference (p<0.01).</p

Flow cytometric analysis of CFTR expression in neutrophils and phagosomes.

<p><b>A</b>) CFTR staining in neutrophils. Purified peripheral-blood neutrophils were immunostained for intracellular CFTR using the monoclonal CFTR antibody (mAb #660). Representative dot plots show that the neutrophils from Myeloid-Cftr−/− mice express CFTR (93%), and the cells from the control Cftr^fl10 mice are 89% positive for CFTR. In contrast, the isotype control antibody shows little background staining. <b>B</b>) CFTR staining in phagosomes. Latex beads (3 µm) were phagocytosed by neutrophils from the control Cftr^fl10 mice or the Myeloid-Cftr−/− mice. Phagosomes released by cell homogenization were immunostained with the same anti-CFTR antibody. The phagosomal population was gated by flow cytometry. Only the phagosomes from the control Cftr^fl10 neutrophils show significant staining for CFTR.</p

Abnormal profile of lung immune cells in Myeloid-Cftr−/− mice in response to lung PsA infection.

<p>Myeloid-Cftr−/− mice and Cftr^fl10 control mice were intubated intratracheally with the agarose-embedded PsA at the sub-lethal dose (1×10⁶ cfu). BAL cells were collected and evaluated for differentials by cytospin and Giemsa staining. At Day 2 after PsA infection, neutrophils were the main constituent of the BAL cells (∼85%) in the lungs of both control mice and myeloid CF mice. In contrast, at Days 3 and 4 neutrophil percent in the Cftr^fl10 control lungs reduced to ∼15% and ∼7%, respectively. However, the percentage of neutrophils in the Myeloid-Cftr−/− lungs dropped only to ∼52% at Day 3 and ∼39% at Day 4, which still remained dominant. Asterisks indicate statistically significant differences by Student’s t-test (n = 4, p<0.01).</p