Transepithelial migration of neutrophils in response to leukotriene B₄ is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade

The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B₄ (LTB₄) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB₄ induces transepithelial migration of neutrophils. We found that LTB₄ induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB₄ receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB₄-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB₄ signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB₄ led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB₄ was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB₄ receptor antagonist.