The underlying infl ammation present in chronic airway diseases is orchestrated by increased expression of CC chemokines that selectively recruit leukocyte populations into the pulmonary system. Human CCL26 signals through CC chemokine receptor 3 (CCR3), is dramatically upregulated in challenged asthmatics, and stimulates recruitment of eosinophils (EOSs) and other leukocytes. CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of chemokines in alveolar type II cells. Utilizing the A549 alveolar type II epithelial cell culture model, we carried out studies to test the hypothesis that CCL26-siRNA treatment of these cells would ameliorate Th2-driven release of the eotaxins and other CCR3 ligands that would, in turn, decrease recruitment and activation of EOSs. Results demonstrate that CCL26-siRNA treatments decreased interleukin-4-induced CCL26 and CCL24 expression by > 70%. CCL26-directed small-interfering RNA (siRNA) treatments signifi cantly decreased release of CCL5 (RANTES), CCL15 (MIP-1δ), CCL8 (MCP-2), and CCL13 (MCP-4). In bioactivity assays it was shown that EOS migration and activation were reduced up to 80% and 90%, respectively, when exposed to supernatants of CCL26-siRNA-treated cells. These results provide evidence that CCL26 may be an appropriate target for development of new therapeutic agents designed to alleviate the underlying infl ammation associated with chronic diseases of the airways.
The underlying inflammation present in chronic airway diseases is orchestrated by increased secretion of CC and CXC chemokines that selectively recruit the leukocyte populations into the pulmonary system. Human chemokines, eotaxins (CCL11 and CCL26), RANTES, and interleukin (IL)-8, are dramatically upregulated through G-protein receptors in cell inflammation, including human asthma. In previous studies, a series of new glucocorticoid antedrugs (GCAs) were synthesized as derivatives of isoxazoline and oxime, and their pharmacological properties based on the antedrug concepts were evaluated. Utilizing both human airway epithelium (HAE) and eosinophil (EOS) cell culture models, we carried out studies to test the hypothesis that new GCA cell treatment would ameliorate Th-1/Th-2-driven secretion of these asthmatic biomarkers, eotaxins (CCL11 and CCL26), RANTES, and IL-8 chemokines, that would in turn decrease recruitment, proliferation, and activation of EOS cells. Results demonstrate that isoxazoline and oxime derivatives exhibit concentration-dependent inhibition, and specifically the compound No. 7 decreases significantly the secretion of eotaxins, RANTES, and IL-8 in cytokine-stimulated HAE cells. It was shown that EOS proliferation and activation were reduced considerably, and cell apoptosis occurred when exposed to nonfluorinated isoxazoline derivatives. These results provide evidence that concentration and structural manipulation of GCAs could increase the anti-inflammatory potency in treatment of chronic diseases, including asthma.
Trafficking and activation of cells in diseases such as asthma are, in part, modulated by eotaxin‐2 (CCL24) and eotaxin‐3 (CCL26) chemokines that signal via CCR3. Short‐interfering RNAs (siRNA) are novel tools to inhibit gene functions in human diseases. In this context, we hypothesized that alveolar type II cells can be transfected with siRNAs targeting CCR3 (CCR3‐siRNA) which will down‐regulate the receptor and decrease synthesis and release of its ligands CCL24 and CCL26. To test our hypothesis, human A549 alveolar type II epithelium‐like cells were transfected with four separate and combined CCR3‐siRNA duplexes and effects on CCR3, CCL26 and CCL24 were studied. FACS analysis showed decreased cell surface CCR3. Densitometry of lysate immunoblots indicated up to 75–84% reduction in CCR3. RT‐PCR showed an 80% decrease in CCR3 mRNA. IL‐4 stimulated CCL26 release (959 ± 201 pg/ml) was reduced up to 65% and constitutive CCL24 release (285 ± 64 pg/ml) was reduced by 80% in CCR3‐siRNA treated cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced up to 80% when treated with supernatants of A549 cells pretreated with CCR3‐siRNAs. These findings suggest that CCR3‐siRNA treatment of alveolar type II cells may be useful for development of novel therapies for controlling airway inflammation. Support for this research was provided in part by NIH grants RR08111 and RR03020.
Antedrug design represents a new approach projected to create safer drugs by inactivating the metabolites after biotransformation. Our primary objective is to synthesis a new group of corticosteroids that have an anti‐asthmatic and anti‐inflammatory purpose without systemic adverse effects.A new antedrugs isoxazoline derivative is synthesized from prednisolone. Various concentrations of isoxazoline derivatives synthesis was tested by ELISA in vitro: human 549 alveolar epithelium and leukocyte macrophage cells after stimulation with cytomix‐induced proinflammatory molecular release and in vivo: receptor‐binding assay using rat lung and liver cytosol. The metabolism of biotransformation was studied in rat plasma using HPLC technique.Most of isoxazoline derivatives showed high binding affinities to the glucocorticoid receptors; five times more potent than prednisolone and significant inhibitory effects of the antedrugs on the nitric oxide (NO) and other proinflammatory cytokines such IL8 cell release. The metabolism study in rat plasma showed that a new isoxazoline with 21‐acetyl groups were hydrolyzed rapidly to inactive metabolites, with half lives 5 minutes.These results suggest that isoxazolines derivatives compared to conventional steroids improve topical anti‐inflammatory activity without concomitant increase in adverse systemic activity. Support by NIH grants S06 GM08111
Trafficking and inflammation in airway diseases are, in part, modulated by members of the CC chemokine family, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), which transduce signals through their CCR3 receptor. In this context, we hypothesized that transfecting alveolar type II epithelial cells with CCR3-targeted siRNA or antisense (AS-ODN) sequences will downregulate cellular synthesis and release of the primary CCR3 ligands CCL26 and CCL24 and will modulate other CCR3 ligands. The human A549 alveolar type II epithelium-like cell culture model was used for transfection and subsequent effects on CCR3 agonists. siRNAs were particularly effective. PCR showed a 60-80% decrease in mRNA and immunoblots showed up to 75-84% reduction of CCR3 in siRNA treated cells. CCR3-siRNA treatments reduced IL-4 stimulated CCL26 release and constitutive CCL24 release by 65% and 80%, respectively. Release of four additional CCR3 agonists RANTES, MCP-2, MCP-3 and MCP-4 was also significantly reduced by CCR3-siRNA treatments of the alveolar type II cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced when eosinophils were treated with supernatants of A549 cells pretreated with CCR3-targeted siRNAs or AS-ODNs. Collectively, the data suggest that post-transcriptional regulation of CCR3 receptors may be a potential therapeutic approach for interrupting proinflammatory signaling.
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