interleukin-8 and 1-10-phenanthroline

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.

Topics: Anti-Infective Agents; Chemokines; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; HeLa Cells; Humans; Hydrogen Peroxide; Interleukin-8; Iron; Oxygen; Phenanthrolines; Reactive Oxygen Species; Signal Transduction

The neutrophil-specific G-protein-coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-alpha-induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF-alpha was most dramatically inhibited by metalloproteinase inhibitors; 1, 10-phenanthroline and EDTA significantly attenuated LPS- and TNF-alpha-induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-alpha-stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF-alpha inhibited IL-8 receptor-mediated calcium mobilization and IL-8-directed neutrophil chemotaxis, both 1, 10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8-mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

Topics: Antigens, CD; Apoptosis; Calcium Signaling; Chemotaxis, Leukocyte; Dexamethasone; Down-Regulation; Edetic Acid; Endocytosis; GTP-Binding Proteins; Humans; Interleukin-8; Leucine; Leukocytes; Lipopolysaccharides; Metalloendopeptidases; Microscopy, Confocal; Phenanthrolines; Protease Inhibitors; Receptors, Chemokine; Receptors, Interleukin; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Tumor Necrosis Factor-alpha