Differential Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Interferon gamma in Mast Cells and Epithelial Cells

Peer reviewed: YesNRC publication: N

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma.

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied.We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry.Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations.PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma

Regulation of Cl− secretion by α2-adrenergic receptors in mouse colonic epithelium

Previous studies have shown that α2 adrenoceptor (α2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of α2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that α2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that α2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 μg ml−1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+–K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 μm). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of α2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that α2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by α2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers

PAR-2 mRNA expression in whole blood of patients with asthma.

<p>(A) PAR-2 mRNA expression in mild/moderate (n = 16) and severe (n = 6) asthmatics. (B) correlation of PAR-2 mRNA expression with percentage of monocytes in peripheral blood. (C) total ICS dose and (D) percentage of FEV₁ predicted in the whole population (n = 22).</p

Asthma exacerbations and CD14⁺⁺CD16⁺PAR-2⁺ expression.

<p>(A) Proportion of CD14⁺⁺CD16⁺PAR-2⁺ cells in peripheral blood was correlated with the total daily dose of ICS in the whole population. (B) Percentage of CD14⁺⁺CD16⁺PAR-2⁺ monocytes in peripheral blood in asthmatics that did not have recent exacerbations (NAE) (n = 26) compared to asthmatics with recent exacerbations (AE) (n = 10). Correlation of the “% of CD14⁺⁺CD16⁺PAR-2⁺ monocytes in peripheral blood” (C) percentage predicted FEV₁ and (D) “% of CRTh2⁺CD4⁺” in peripheral blood in subjects with asthma exacerbations (n = 10).</p

ROC curve of “% of CD14⁺⁺CD16⁺PAR-2⁺ monocytes” in peripheral blood used to identify patients with severe asthma.

<p>Sensitivity and specificity were calculated according to the optimal cutoffs using R software and AUC with a 95% confidence interval is shown.</p

PAR-2 expression on monocytes and severe asthma

<p><b>A.</b> Gating strategy to study PAR-2 expression on peripheral blood monocytes. B-C. Percentage of CD14⁺⁺CD16⁺ (B) and CD14⁺⁺CD16^- (C) monocytes in the peripheral blood of severe asthmatics compared to mild/moderate asthmatics. D-E. PAR-2 expression on CD14⁺⁺CD16⁺ (D) and CD14⁺⁺CD16^- (E) monocytes in patients with mild/moderate and severe asthma. F-G. Percentage of CD14⁺⁺CD16⁺PAR-2⁺ (F) and CD14⁺⁺CD16^-PAR-2⁺ (G) monocytes in peripheral blood of patients with mild/moderate and severe asthma. H-I. PAR-2 MFI on CD14⁺⁺CD16⁺ (H) and CD14⁺⁺CD16^- (I) monocytes from patients with mild/moderate and severe asthma. Data is presented as boxplots (n = 24 for mild/moderate and n = 12 for severe asthma). Statistical significance was assessed by Mann-Whitney rank sum test, with <i>P</i><0.05 considered significant.</p