SiRNA silencing of eotaxin‐3 (CCL26) modulates gene expression of eotaxin‐2 (CCL24) and CCR3 in alveolar type II cells

Abstract

Chronic diseases of the airway are characterized by an underlying inflammation. The CC chemokines CCL26 and CCL24 recruit and activate eosinophils (EOS) which participate in the airway inflammation. Our previous work demonstrated that CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of cytokines and chemokines in alveolar type II cells. Thus, we hypothesized that treatment of these cells with CCL26‐siRNA may decrease cytokine‐induced release of the eotaxins and suppress EOS activation. A549 alveolar type II cells were transfected with four CCL26‐siRNA duplexes then stimulated with IL‐4. ELISA results indicated >90% inhibition of IL‐4‐stimulated release of CCL26 (789±33.4 pg/ml) and CCL24 (206±2.31 pg/ml) in CCL26‐siRNA treated A549 cells. Densitometry results indicated a 64–71% decrease in CCR3 protein expression in siRNA treated cells and a >88% decrease in CCL26 mRNA as indicated by dotblots and RT‐PCR, respectively. Effects of siRNA treatments on EOS activation were then tested. Results indicated that CCL26‐siRNA treatment of alveolar type II cells reduced EOS superoxide anion generation by 70–90%. These findings demonstrate that siRNA technology may provide a new strategy for post‐transcriptional control of eotaxin gene expression, thereby ameliorating inflammation occurring in airway diseases. Supported in part by NIH grants RR08111 and RR03020.