Original Contributions
The role of oxidative stress in rhinovirus induced elaboration of IL-8 by respiratory epithelial cells

https://doi.org/10.1016/S0891-5849(98)00233-0Get rights and content

Abstract

A direct correlation has been reported between the severity of symptoms associated with rhinovirus infection and the concentration of interleukin-8 in nasal secretions. The purpose of these studies was to examine the mechanism of rhinovirus-induced IL-8 elaboration. Rhinovirus infection induced oxidative stress in Beas-2b cells and the concentration of H2O2 in supernatant media from rhinovirus challenged cells was 12.5 ± 6.1 μM 1 h after challenge compared to 0.7 ± 0.3 μM in supernatant from control cells. N-acetyl cysteine inhibited RV-induced NF-κB activation and IL-8 elaboration. IL-8 concentrations were 36 ± 2 pg/ml and 10 ± 1 pg/ml 6 h after virus challenge in untreated and NAC-treated (30 mM NAC) cells, respectively. Despite the effects of NAC on IL-8 elaboration and NF-κB activation, RV stimulated increases in supernatant H2O2 were not altered by NAC. These data suggest that RV stimulation of IL-8 in respiratory epithelium is mediated through production of oxidative species and the subsequent activation of NF-κB.

Introduction

The rhinoviruses are an important cause of common cold symptoms. Infection with these viruses contributes to the development of sinusitis and otitis media and has been identified as an important cause of exacerbations of asthma in children. The mechanism by which rhinoviruses cause illness is poorly understood. The observation that polymorphonuclear leukocytes (PMNs) infiltrate the nasal mucosa and are expelled in the nasal secretions of subjects who are infected and ill but not those who are infected but asymptomatic suggests that the host inflammatory response may play a role in common cold pathogenesis [1], [2]. Interleukin-8 (IL-8), a potent chemoattractant for PMNs is found in increased concentration in the nasal secretions of human volunteers with symptomatic rhinovirus colds [3], [4]. Furthermore, there is a direct correlation between the severity of nasal obstruction, rhinorrhea and total symptoms and the concentration of IL-8 in the nasal secretions [4]. These data suggest that the inflammatory response to rhinovirus infection and the subsequent common cold symptoms may be mediated in part by IL-8.

Activation of NF-κB appears to be an essential step in the signal transduction pathway that results in elaboration of IL-8 [5]. Although a variety of different stimuli may cause activation of NF-κB, reactive oxygen species (ROS) seem to be a common intermediate in this activation [6]. The purpose of these studies was to examine the role of oxidant stress in general and hydrogen peroxide in particular in the activation of NF-κB and the elaboration of IL-8 by human respiratory cell lines in response to rhinovirus infection. The studies reported in this manuscript show that oxidative stress-induced signaling events are involved in the activation of NF-κB and the elaboration of IL-8 following rhinovirus challenge and that these signaling events can be blocked by N-acetyl cysteine an anti-oxidant. These observations may suggest a potential new approach to the development of therapeutic interventions for rhinovirus induced common cold symptoms.

Section snippets

Cell culture

All experiments were done in human respiratory cell lines maintained at 37°C in 5% CO2. Human embryonic lung fibroblast cells (MRC-5, Biowhittaker, Walkersville, MD, USA) were grown in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% fetal calf serum , 5 u/ml penicillin G sodium, and 5 μg/ml streptomycin. Cells were used for experiments at passage 21–26 within 2 days of the time the monolayers became confluent. Human bronchial epithelial cells (Beas-2b, ATCC, Rockville, MD, USA)

Stimulation of IL-8 by H2O2

Beas-2b and MRC-5 cells stimulated with H2O2 elaborated IL-8 in a dose-dependent manner. Six h after Beas-2b cells were challenged with 0.5 mM H2O2 the supernatant media contained 176 ± 62 pg/ml of IL-8 compared to 10 ± 3 pg/ml in media from control cells (Fig. 1). Similarly, MRC-5 cells stimulated with 0.3 mM H2O2 elaborated 507 ± 45 pg/ml of IL-8 after 6 h compared to 322 ± 40 pg/ml elaborated by control cells. Higher concentrations of H2O2 were cytotoxic in both cell lines as determined by

Discussion

Our studies demonstrate that rhinovirus infection of human respiratory epithelial cells results in increased production of hydrogen peroxide and oxidative stress. Challenge of the cells with either rhinovirus or hydrogen peroxide caused translocation of NF-κB to the nucleus and elaboration of IL-8. Pre-treatment of the cells with NAC, a potent antioxidant, inhibited rhinovirus and hydrogen peroxide mediated NF-κB activation and IL-8 elaboration, however, NAC did not inhibit the increased

Acknowledgements

Acknowledgement — This work was supported in part by NIH grants NS-22576 and NS-34741 (Dr. Singh).

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