E1A expression dysregulates IL-8 production and suppresses IL-6 production by lung epithelial cells.

Arjen van den Berg,Henk M Jansen,René Lutter,Mieke Snoek

doi:10.1186/1465-9921-6-111

Arjen van den Berg, Henk M Jansen + Show 2 more

Open Access

https://doi.org/10.1186/1465-9921-6-111

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Abstract

BackgroundThe adenoviral protein E1A has been proposed to play a role in the pathophysiology of COPD, in particular by increasing IL-8 gene transcription of lung epithelial cells in response to cigarette smoke-constituents such as LPS. As IL-8 production is also under tight post-transcriptional control, we planned to study whether E1A affected IL-8 production post-transcriptionally. The production of IL-6 by E1A-positive cells had not been addressed and was studied in parallel. Based on our previous work into the regulation of IL-8 and IL-6 production in airway epithelial cells, we used the lung epithelial-like cell line NCI-H292 to generate stable transfectants expressing either E1A and/or E1B, which is known to frequently co-integrate with E1A. We analyzed IL-8 and IL-6 production and the underlying regulatory processes in response to LPS and TNF-α.MethodsStable transfectants were generated and characterized with immunohistochemistry, western blot and flow cytometry. IL-8 and IL-6 protein production was measured by ELISA. Levels of IL-8 and IL-6 mRNA were measured using specific radiolabeled probes. EMSA was used to assess transcriptional activation of relevant transcription factors. Post-transcriptional regulation of mRNA half-life was measured by Actinomycin D chase experiments.ResultsMost of the sixteen E1A-expressing transfectants showed suppression of IL-6 production, indicative of biologically active E1A. Significant but no uniform effects on IL-8 production, nor on transcriptional and post-transcriptional regulation of IL-8 production, were observed in the panel of E1A-expressing transfectants. E1B expression exerted similar effects as E1A on IL-8 production.ConclusionOur results indicate that integration of adenoviral DNA and expression of E1A and E1B can either increase or decrease IL-8 production. Furthermore, we conclude that expression of E1A suppresses IL-6 production. These findings question the unique role of E1A protein in the pathophysiology of COPD, but do not exclude a role for adenoviral E1A/E1B DNA in modulating inflammatory responses nor in the pathogenesis of COPD.

Highlights

The adenoviral protein E1A has been proposed to play a role in the pathophysiology of Chronic obstructive pulmonary disease (COPD), in particular by increasing IL-8 gene transcription of lung epithelial cells in response to cigarette smoke-constituents such as LPS
As expression of E1A leads to apoptosis in non-ras-transformed cells [17,18], it is likely that NCI-H292 cells expressing E1A protein underwent apoptosis
This suggests that the larger response range in AB- and Bclones is caused by the presence of E1A and E1B DNA, part of the response range appears due to biological variation of the motherline and/or results from the procedure of subcloning. To further test the latter we cloned an earlier derived subclone and tested TNF-α- and LPS-induced responses from 11 derived sub-subclones. This time, we found a 4-fold difference between clones in their maximal IL-6 and IL-8 responses, which indicates that there is some variation in the NCI-H292 motherline

Summary

Introduction

The adenoviral protein E1A has been proposed to play a role in the pathophysiology of COPD, in particular by increasing IL-8 gene transcription of lung epithelial cells in response to cigarette smoke-constituents such as LPS. Hogg and coworkers have put forward the concept that the presence of the adenoviral E1A DNA and protein in airway structural cells, leading to enhanced IL-8 production in response to endotoxin exposure, may be related to the development of COPD. First they showed by PCR analysis that lung tissue from COPD patients contained more E1A DNA than lung tissue from matched non-COPD smokers [2]. The in vivo relevance of E1A expression was illustrated with a guinea pig model, showing an enhanced inflammatory response to cigarette smoke in animals with lung tissue containing E1A DNA [8]

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