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Respiratory syncytial virus and TNF alpha induction of chemokine gene expression involves differential activation of Rel A and NF-kappa B1 - PubMed

  • ️Tue Jan 01 2002

Comparative Study

Respiratory syncytial virus and TNF alpha induction of chemokine gene expression involves differential activation of Rel A and NF-kappa B1

Laura R Carpenter et al. BMC Infect Dis. 2002.

Abstract

Background: Respiratory syncytial virus (RSV) infection of airway epithelial cells stimulates the expression and secretion of a variety of cytokines including the chemotactic cytokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated upon activation, normal T cell expressed and secreted). Chemokines are important chemoattractants for the recruitment of distinct sets of leukocytes to airway sites of inflammation.

Results: We have shown previously that chemokine expression is regulated in airway epithelial cells (A549) in a stimulus-specific manner in part through the redox-responsive transcription factors AP-1 and NF-kappaB. In this study, we examined the NF-kappaB-mediated effects of RSV and the proinflammatory cytokine TNFalpha on the induction of IL-8, MCP-1 and RANTES chemokine gene expression in A549 epithelial cells. The results demonstrate that RSV induces chemokine expression with distinct kinetics that is associated with a specific pattern of NF-kappaB binding activity. This distinction was further demonstrated by the differential effects of the NF-kappaB inhibitors dexamethasone (DEX) and N-acetyl-L-cysteine (NAC). NAC preferentially inhibited RSV induced chemokine expression, whereas DEX preferentially inhibited TNFalpha induced chemokine expression. DNA binding studies using NF-kappaB subunit specific binding ELISA demonstrated that RSV and TNFalpha induced different NF-kappaB binding complexes containing Rel A (p65) and NF-kappaB1 (p50). Both TNFalpha and RSV strongly induced Rel A the activation subunit of NF-kappaB, whereas only TNFalpha was able to substantially induce the p50 subunit. Consistent with the expression studies, RSV but not TNFalpha induction of Rel A and p50 were markedly inhibited by NAC, providing a mechanism by which TNFalpha and RSV can differentially activate chemokine gene expression via NF-kappaB.

Conclusions: These data suggest that RSV induction of chemokine gene expression, in contrast to TNFalpha, involves redox-sensitive NF-kappaB complexes containing predominantly Rel A.

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Figures

Figure 1
Figure 1

RSV induces chemokine protein secretion from A549 cells. 2 × 105 A549 cells were infected by RSV (MOI = 3) for 24 hr and supernatants assessed for IL-8, MCP-1 and RANTES by chemokine specific ELISA. The histogram shows the mean pg/ml of protein detected from three independent experiments. The error bars show the standard deviation from the mean.

Figure 2
Figure 2

Chemokine mRNA expression induced by RSV and TNFα. 2 × 107 A549 cells were either infected by RSV (MOI = 1) or stimulated with 100 ng/ml TNF-α. Total RNA was isolated at 1,2, 4, 8, and 24 h and assessed for chemokine expression by RNase protection. Chemokine expression was quantified by densitometry as described in the Methods. The graphs show the time course of chemokine mRNA after normalizing to GAPDH (lowest band in the gel). Shown is representative of 3 independent experiments.

Figure 3
Figure 3

Chemokine mRNA inhibition in A549 cells. 2 × 107 A549 cells were pretreated with 5 mM N-acetyl-L-cysteine (NAC) or 500 nM dexamethasone (DEX). After 1 hr the pretreated cells were either infected with RSV (MOI = 1) or stimulated with TNFα (100 ng/ml) for 24 hr. The graphs show the percent inhibition for each chemokine mRNA after normalization to GAPDH. Shown is representative of two independent experiments.

Figure 4
Figure 4

Induction of Rel A (p65) and NF-κB1 (p50) by RSV and TNFα is differentially inhibited by NAC and DEX. 2 × 107 A549 cells were pretreated with 5 mM N-acetyl-L-cysteine (NAC) or 500 nM dexamethasone (DEX). After 1 hr the pretreated cells were either stimulated with TNFα (100 ng/ml) or infected with RSV (MOI = 1) for 2 hr. Nuclear extracts (3 μg) were prepared and assessed for Rel A (p65, solid bars) or NF-κB1 (p50, open bars) binding activity using a Trans-AM™ transcription factor assay kit from Active Motif (Carlsbad, CA) as per the manufacturer's instructions. Graph shows the NF-κB activation results (OD450nm) from A549 cells stimulated with TNFα (A) or infected with RSV (B). An excess of either wildtype (wt) or mutant (mut) NF-κB oligonucleotide provided by the kit was included in the binding reactions to demonstrate NF-κB binding specificity. Note that NAC and DEX inhibits RSV induced p65 and p50 whereas the inhibitors had no effect on TNFα induced NF-κB binding activity.

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