Regulation of chemokine production via oxidative pathway in HeLa cells

Abstract

Inflammation is associated with disease progression and, by largely unknown mechanisms, has been said to drive oncogenesis. At inflamed sites, neutrophils deploy a potent antimicrobial arsenal that includes proteinases, antimicrobial peptides, and ROS. Reactive oxygen species (ROSs) induce chemokines. In the present study, the concentrations of IL-8 in culture supernatants of HeLa cells treated with ROS were determined by enzyme-linked immunosorbent assay. We used o-phenanthroline to deplete Fe(2+) in order to investigate the mechanisms through which ROSs induce IL-8 secretion in our system. The iron chelator o-phenanthroline effectively inhibited H(2)O(2)-induced ERK2 activation. Enzyme-linked immunosorbent assays showed that IL-8 protein secretion was elevated in ROS-treated HeLa cells. When Fe(2+) was removed from these cells, IL-8 secretion was inhibited. Collectively, these results indicate that Fe(2+)-mediated Erk pathway activation is an important signal transduction pathway in ROS-induced IL-8 secretion in epithelial cells.

Figure 1

Time course of the activation of Erk and IL-8 production by H₂O₂. (a) Activated Erk: phosphorylated Erk1/2 induced by 0.25 mM H₂O₂ at the indicated time points of treatment. (b) Expression levels of IL-8 mRNA induced by incubation with 0.25 mM at the indicated time points.

Figure 2

Erk controls H₂O₂-induced IL-8 expression in HeLa cells. The inhibitory effect of PD98059 on H₂O₂-induced expression (0.25 mM) of IL-8 mRNA ((a) real-time PCR) and protein secretion ((b) ELISA). Data points are means ± s.e.m. *P<.01 (n = 3).

Figure 3

The role of free radicals in H₂O₂-mediated Erk2 activation and IL-8 production. (a) Mannitol (100 mM), o-phenanthroline (0.2 mM), or EGTA (2.5 mM) was added 45 minutes before direct addition of H₂O₂, and cells were harvested 15 minutes later for the analysis of p-Erk1/2 activity for the western blot. The inhibitors alone did not activate Erk1/2. (b) The inhibitory effects of o-phenanthroline on H₂O₂-induced expression (0.25 mM) of IL-8 protein secretion (ELISA) *P<.01 (n = 3).