Low grade rhinovirus infection induces a prolonged release of IL-8 in pulmonary epithelium.

Rhinoviruses are important respiratory pathogens implicated in asthma exacerbations. The mechanisms by which rhinoviruses trigger inflammatory responses in the lower airway are poorly understood, in particular their ability to infect the lower airway. Bronchial inflammatory cell (lymphocyte and eosinophil) recruitment has been demonstrated. IL-8 is a potent proinflammatory chemokine that is chemotactic for neutrophils, lymphocytes, eosinophils, and monocytes and may be important in the pathogenesis of virus-induced asthma. Increased levels of IL-8 have been found in nasal samples in natural and experimental rhinovirus infections. In these studies we therefore examine the ability of rhinovirus to infect a transformed lower airway epithelial cell line (A549) and to induce IL-8 protein release and mRNA induction. We observed that rhinovirus type 9 is able to undergo full viral replication in A549 cells, and peak viral titers were found 24 h after inoculation. Rhinovirus infection induced a dose- and time-dependent IL-8 release up to 5 days after infection and an increase in IL-8 mRNA expression that was maximal between 3 and 24 h after infection. UV inactivation of the virus completely inhibited replication, but only reduced IL-8 protein production and mRNA induction by half, while prevention of virus-receptor binding completely inhibited virus-induced IL-8 release, suggesting that part of the observed effects was due to viral replication and part was due to virus-receptor binding. These studies demonstrate that rhinoviruses are capable of infecting a pulmonary epithelial cell line and inducing IL-8 release. These findings may be important in understanding the pathogenesis of rhinovirus-induced asthma exacerbations.

Activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-kappaB in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium.

The role of "oxidant-sensitive" transcription factors activator protein (AP)-1, nuclear factor (NF)-kappaB, and NF-IL6 in respiratory syncytial virus (RSV)-induced interleukin (IL)-8 gene expression in A549 epithelial cells was evaluated. RSV infection resulted in increased binding of each of these transcription factors. Transfection of A549 cells with plasmids containing serial truncations of the 5'-flanking region of the IL-8 gene revealed a positive cooperative effect of the binding sites for AP-1 and NF-kappaB. Mutation of either region markedly diminished responsiveness of the promoter to RSV. Mutation of the NF-IL6 site had minimal effect in the presence of intact binding sites for NF-kappaB and AP-1. The antioxidants NAC (N-acetylcysteine), DMSO, and DMPO (5,5-dimethyl-1-pyrroline N-oxide) did not inhibit RSV-induced binding of NF-kappaB; however, binding of AP-1 and NF-IL6 was inhibited. These observations suggest that AP-1 may be the preferred transcription factor (over NF-IL6) for cooperative interaction with NF-kappaB in RSV-induced IL-8 production.

Amiloride inhibits cytokine production in epithelium infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is an important respiratory pathogen in infants and children. RSV preferentially infects airway epithelium and causes local production of inflammatory cytokines. Ribavirin, the only specific agent available for treatment of RSV infection, has limited effectiveness. There are few data regarding the ability of drugs to modulate the inflammatory response of epithelium infected with RSV. This study evaluated the effect of amiloride and ribavirin on cytokine production by RSV-infected epithelium. We observed a dose-dependent reduction in interleukin (IL)-8 protein release with both amiloride and ribavirin in RSV-infected A549 epithelial cells. Peak effects were observed at concentrations of 200 microM amiloride and 60 microM ribavirin. Both amiloride and ribavirin inhibited IL-8 mRNA induction. Pretreatment with either agent was not required to inhibit IL-8 release. Both drugs also inhibited IL-6 release. However, unlike ribavirin, amiloride did not inhibit viral replication or infection. Amiloride also inhibited IL-8 release from A549 cells stimulated with IL-1 or tumor necrosis factor. Amiloride similarly inhibited IL-8 protein release from primary human airway epithelium infected with RSV. These data demonstrate that both amiloride and ribavirin inhibit cytokine production in RSV-infected airway epithelium. These results suggest amiloride, as well as ribavirin, may be useful as a therapeutic agent in RSV infections.

Induction of interleukin (IL)-8 gene expression by respiratory syncytial virus involves activation of nuclear factor (NF)-kappa B and NF-IL-6.

Respiratory syncytial virus (RSV) preferentially infects respiratory epithelium and is an important cause of lower respiratory tract infections in young children. RSV induces the production of interleukin (IL)-8 in airway epithelial cells; however, the mechanism of this induction is not known. To define the mechanism by which RSV induces IL-8 gene activation, A549 epithelial cells were transfected with plasmids containing serial deletions of the 5'-flanking region of the IL-8 gene and then exposed to RSV for 24 h. A positive cooperative effect of the binding sites for the transcription factors, nuclear factor (NF)-kappa B and NF-IL-6, was observed. Mutations in either region abates responsiveness of the promoter to RSV infection. RSV also increases activation of the NF-kappa B and NF-IL-6 transcription factors. These data suggest that RSV may increase IL-8 production in airway epithelium partly via activation of the transcription factors NF-kappa B and NF-IL-6.

Oxidant tone regulates IL-8 production in epithelium infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway, and causes a local inflammatory response. Although it has been previously demonstrated that RSV-infected airway epithelial produce cytokines, including interleukin-8 (IL-8), which contributes to the inflammatory response, the regulation of this effect of RSV is unknown. To further characterize the mechanisms by which RSV infection triggers release of IL-8, we first exposed cultured A549 cells to RSV, and measured IL-8 release via enzyme-linked immunosorbent assays (ELISA), and IL-8 messenger RNA (mRNA) induction via Northern blot analysis. We observed a dose- and time-dependent release of IL-8 in response to RSV. The optimal dose of RSV was 10(4) TCID50/ml, and maximal release of IL-8 was measured at 72 to 96 h after infection. RSV induced a biphasic (early and late) increase in IL-8 mRNA. The early phase was independent of viral infection, whereas the more pronounced late phase required the presence of live virus and infection of the epithelium. Partial (< 50%) cytopathic effects were noted at 48 h and progressed to 75% at 96 h. The monolayer was still intact at 96 h. Inhibitors of nitric oxide, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and aminoguanidine had no effect on IL-8 release or IL-8 mRNA induction. We did, however, demonstrate a dose-dependent decrease in IL-8 release and IL-8 mRNA induction in RSV-infected epithelial treated with the antioxidants dimethyl sulfoxide (DMSO) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Peak effects were noted at a concentration of 2% DMSO and 50 microM DMPO. The antioxidants did not inhibit viral replication or infection. This data suggest that RSV-induced IL-8 production in airway epithelium is mediated via changes in oxidant tone. The data also suggest a potential therapeutic role for antioxidants in RSV infections.