Abstract
Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.
Highlights
The potent neutrophil chemokine interleukin (IL)-8 [1] is produced and secreted in the airways as part of innate immune responses to inhaled ‘‘invaders’’
Pretreatment with the epidermal growth factor receptor (EGFR)-selective inhibitor AG1478 prevented the IL-8 production induced by CFTR-172 completely (Fig. 1B), whereas pretreatment with the platelet-derived growth factor receptor inhibitor AG1295 had no significant effect on IL-8 production (Fig. 1B)
Pretreatment with an EGFR neutralizing antibody inhibited the IL-8 production induced by CFTR-172 completely (Fig. 1B)
Summary
The potent neutrophil chemokine interleukin (IL)-8 [1] is produced and secreted in the airways as part of innate immune responses to inhaled ‘‘invaders’’ (eg, bacteria, viruses, cigarette smoke). There is growing evidence that exaggerated IL-8 production may be an intrinsic property of airway epithelial cells lacking normal CFTR. Airway epithelial cells that contain mutant CFTR have been shown to produce more IL-8 in response to bacterial products [10,11,12] and to IL-1 [13], and to produce more IL-8 in the constitutive state [10,11,14,15,16], than isogenic cells corrected with wild-type CFTR. Treatment of airway epithelial cells that contain normal CFTR with CFTRselective inhibitors has been shown to induce IL-8 production [17,18,19]. Together, these findings suggest that loss of normal CFTR function exaggerates airway epithelial IL-8 production
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