Metalloprotease-dependent amphiregulin release mediates tumor necrosis factor-α-induced IL-8 secretion in the human airway epithelial cell line NCI-H292

Tumor necrosis factor-α (TNF-α) is a potent multifunctional cytokine that plays a central role in the pathogenesis of many inflammatory diseases. Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. The alveolar macrophage-derived TNF-α initiates lung inflammation through its ability to stimulate IL-8 synthesis in airway epithelial cells. Since recent studies demonstrated that the stimulation of epidermal growth factor receptor (EGFR) could induce IL-8 secretion, the involvement of EGFR in TNF-α-induced IL-8 secretion in airway epithelium-like NCI-H292 cells was investigated in this study. TNF-α and epidermal growth factor (EGF) stimulated IL-8 secretion in a time- and concentration-dependent manner. Inhibition of the EGFR by either an anti-EGFR neutralizing antibody or by its specific inhibitor AG1478 (1 μM) blocked TNF-α-induced IL-8 secretion. In addition, TNF-α stimulated tyrosine phosphorylation of the EGFR within 5 min after stimulation. Further, TNF-α-induced IL-8 secretion was completely inhibited by the neutralizing antibody against amphiregulin (AR), an EGFR ligand, suggesting that TNF-α-induced IL-8 secretion was mediated by the AR-EGFR pathway. Furthermore, TNF-α stimulated the release of AR in a concentration-dependent manner. Finally, both AR and IL-8 release-induced by TNF-α were eliminated by pretreatment with either GM6001, a broad-spectrum inhibitor for metalloprotease, or TAPI-1, relatively selective inhibitor for TNF-α converting enzyme (TACE). These findings indicate that metalloprotease-mediated AR shedding and subsequent activation of EGFR play a critical role in TNF-α-induced IL-8 secretion from the human airway epithelium-like NCI-H292 cells, and that TACE is one of the most possible candidates for metalloprotease responsible for TNF-α-induced AR shedding.