Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

https://doi.org/10.1016/j.taap.2009.11.011Get rights and content

Abstract

Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

Introduction

Diesel exhaust particles (DEP) are ubiquitously present in ambient air as a major contributor to the fine and ultrafine fraction of particulate matter (PM) in urban areas (Lloyd and Cackette, 2001). DEP contain a carbon core with adsorbed organic compounds such as polycyclic aromatic hydrocarbons and quinones (Wichmann, 2007). Some of these organic compounds and their reactive metabolites are intrinsically electrophilic (Iwamoto et al., 2007) or can generate reactive oxygen species (ROS) through redox reactions (Li et al., 2002, 2003). There is increasing evidence to suggest that the adverse effects of PM exposure are attributable to its oxidative properties (Shinyashiki et al., 2009) and its ability to promote intracellular ROS generation (Li et al., 2003).

Airway epithelial cells are known targets of inhaled DEP and contribute to PM-induced airway inflammation by cytokine and chemokine synthesis. For instance, it has been previously shown that DEP induces the expression of proinflammatory cytokines and chemokines in cultured airway epithelial cells including IL-8, IL-1α, GM-CSF, and Gro-α (Bonvallot et al., 2001, Matsuzaki et al., 2006, Baulig et al., 2007). In agreement with these in vitro findings, enhanced epithelial expression of IL-8, IL-13 and Gro-α was reported in bronchial biopsies obtained from human subjects exposed to DEP (Salvi et al., 2000, Pourazar et al., 2005). The expression of proinflammatory mediators by DEP exposure is controlled at the transcriptional level by a number of signaling pathways that activate transcription factors such as AP-1 and NFkB (Takizawa et al. 1999). Therefore, these studies support the notion that DEP exposure promotes pulmonary inflammation mediated by MAPK signaling, activation of NFkB and AP-1, and leads to the synthesis and release of proinflammatory mediators by airway epithelium.

IL-8 is a potent neutrophil activator and chemotaxin that is secreted by airway epithelial cells and is often used as a biological marker of environmentally induced pulmonary inflammation (Strieter, 2002). In differentiated human nasal epithelial cells cultured under air–liquid interface, IL-8 secretion is highly polarized, resulting in secretion towards the basolateral compartment following DEP exposure (Auger et al., 2006). These data suggest that the response of an intact airway epithelium to DEP could contribute to the local inflammatory process characteristic of PM exposure in vivo. IL-8 expression is increased in airway epithelial cells exposed to ambient PM (Frampton et al., 1999) and the metallic PM components Zn2+ and V4+ (Samet et al., 1998, Kim et al., 2006) as well as to DEP (Salvi et al., 1999Salvi et al., 2000, Mudway et al., 2004), residual oil fly ash (ROFA) particles (Carter et al., 1997) and the gaseous air pollutant ozone (Jaspers et al., 1997a, 1997b) as well as in bronchial biopsies from human subjects exposed to DEP (Salvi et al., 2000, Holgate et al., 2003). Although some information concerning DEP-induced activation of upstream redox-sensitive signaling pathways has been reported (Takizawa et al., 1999, Pourazar et al., 2005), the exact mechanism by which DEP exposure induces IL-8 expression remains unclear. Therefore, because IL-8 expression is induced by a broad spectrum of PM-associated air pollutants both in vitro and in vivo, in the current study, we sought to investigate the upstream mechanisms by which DEP stimulates IL-8 expression in airway epithelial cells.

The IL-8 gene promoter region contains multiple 5′ regulatory elements, including binding sites for NFkB, AP-1, AP-2, AP-3, CCAAT/enhancer binding protein β (C/EBP β), interferon regulatory factor 1, and a glucocorticoid response element (Mukaida et al., 1998, Strieter, 2002). Exposure to a DEP with high organic content (A-DEP) has been previously shown to induce NFkB-dependent IL-8 expression in the human epithelial cell line BEAS-2B (Takizawa et al., 1999). In addition, a different low-organic containing DEP (N-DEP) has also been reported to stimulate IL-8 expression in the same cell type (Singh et al., 2004, Cao et al., 2007a, 2007b), suggesting that DEP induces proinflammatory responses in airway epithelial cells through multiple mechanisms. In the present study, we have examined the effect of exposure to DEP with varying organic content on the expression of IL-8 in human airway epithelial cells. We report here that exposure to DEP with high or low organic content induces IL-8 expression through the activation of distinct transcriptional regulatory pathways. These studies also demonstrate the utility of using a suite of canonical and gene-specific promoter reporter constructs to quantitatively assess the proinflammatory potential of ambient air pollutants in cultured cells.

Section snippets

Preparation of DEP

Preparation of the three DEP used in the current study has been previously described (Tal et al., 2008). Briefly, N-DEP with low organic content was obtained from NIST (NIST SRM 2975; Minneapolis, MN). N-DEP contains 1.5% (wt/wt) extractable organic matter (EOM) by dichloromethane extraction (Stevens et al., 2009). The second sample, Compressor DEP (C-DEP) with intermediate organic content, was generated at the U.S. EPA (RTP, NC) and contains 18.9% (wt/wt) EOM by dichloromethane extraction (

DEP exposure induces IL-8 expression in human airway epithelial cells

To study the effects of DEP exposure on proinflammatory signaling in lung epithelial cells, we used qRT-PCR to measure IL-8 expression in HAEC treated for 4 h with 10 μg/cm2  N-DEP, C-DEP, A-DEP, or CB. IL-8 mRNA levels were normalized to GAPDH mRNA levels and expressed as the fold change over untreated control. As shown in Fig. 1A, DEP treatment induced expression of IL-8 with varying efficacy, with A-DEP exposure resulting in the most robust increase in IL-8 mRNA, followed by N-DEP (23.5 ± 8.0

Discussion

Airway epithelial cells are directly exposed to inhaled DEP, and are a significant source of inflammatory mediators. We have previously reported that exposure of airway epithelial cells to L-DEP induces proinflammatory responses characterized by increased expression of IL-8 and Cox-2 (Cao et al., 2007a, 2007b). However, the signaling events involved and the role of DEP composition in inducing these responses are not clear. The present study shows that exposure to DEP with high and low organic

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank Melanie Jardim for helpful discussions and the critical review of this manuscript. We are grateful to M. Ian Gilmour and David Diaz-Sanchez for providing us with C-DEP and A-DEP, respectively. T.L.T. is a recipient of the Curriculum in Toxicology Training Grant T32 ES007126. Fellowship support for S.H.C. from the U.S. EPA Research Participation Program administered by ORISE is acknowledged. This research was also supported by EPA training grant T829472.

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    Disclaimer: The research described herein has been reviewed by the National Health and Environmental Effects Research Laboratory and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor does mention of trade names constitute endorsement of recommendation for use.

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    Present address: Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, USA.

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