Date of Award

1997

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Ron Turner

Second Advisor

Salvatore Arrigo

Third Advisor

Lucille London

Fourth Advisor

Steven D. London

Fifth Advisor

Steven A. Rosenzweig

Abstract

There is a direct correlation between the severity of common cold symptoms and the concentration of IL-8 in nasal secretions from volunteers with experimental human rhinovirus (hRV) colds. Although these and other data are consistent with the hypothesis that IL-8 is a mediator of common cold symptoms, the mechanism by which IL-8 is elaborated in response to hRV challenge is not understood. This study examined the role of oxidative stress and NF-κB activation in hRV induced IL-8 elaboration. All studies were done in either a transformed respiratory epithelial cell line (Beas-2b) or human lung fibroblasts (MRC-5). Induction of oxidative stress by hRV was demonstrated by fluorescent staining of carbonyl groups, conversion of dichlorofluorescein diacetate (DCFDA) to fluorescein, and H2O2 production. H2O2 concentrations were 4.74 + 2.04 µM and 0.40 + 0.49 µM in supernatants from hRV challenged and control Beas-2b cells, respectively (p<0.01, Wilcoxon sign rank). Treatment of both MRC-5 and Beas-2b cells with exogenous H2O2 resulted in a dose dependent elaboration of IL-8. Antioxidants attenuated IL-8 secretion by viral treated cells. Treatment of Beas-2b and MRC-5 cells with N-acetyl cysteine (NAC) inhibited hRV induced IL-8 elaboration. IL-8 concentrations in supernatants from Beas-2b cells were 35.8 + 2.1 pg/ml and10.0 + 1.0 after virus challenge in untreated and treated cells (30 mM NAC) respectively. In MRC-S cells viral stimulation of IL-8 was reduced from 403.8 + 85.7 pg/ml to 239 + 67.7 pg ml IL-8 (p<0.02, Wilcoxon sign rank) by treatment with 20 mM NAC. The antioxidants, dimethyl sulfoxide (DMSO) and ferulic acid also inhibited viral-induced IL-8 in both cell lines; two other antioxidants, 5'5'-Dimethyl-1-Pyrroline N-Oxide (DMPO) and α-Phenyl-tert-butyl Nitrone (PBN) had little effect on IL-8 elaboration. The production of IL-8 in response to oxidative stress appeared to be mediated by NF-κB. NF-κB was activated in virus challenged Beas-2b cells but not in unchallenged cells and this activation was inhibited by treatment with 20 mM NAC. Activation of NF-κB was also seen in Beas-2b cells following treatment with 1 mM H202. These data suggest that hRV stimulation of IL-8 is mediated through the production of reactive oxygen species (ROS) and their subsequent activation of NF-κB. The antioxidants NAC, DMSO, and ferulate inhibit hRV induced IL-8 elaboration although the precise mechanism of this inhibition has not been determined.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS